Identification of mutations in channelopsin variants having improved light sensitivity and methods of use thereof

ABSTRACT

The invention provides compositions and kits including at least one nucleic acid or polypeptide molecule encoding for a mutant CoChop protein. Methods of the invention include administering a composition comprising a mutant CoChop to a subject to preserve, improve, or restore phototransduction. Preferably, the compositions and methods of the invention are provided to a subject having impaired vision, thereby restoring vision to normal levels.

RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 16/328,916 filed Feb. 27, 2019, now U.S. Pat. No. 11,041,004issued on Jun. 22, 2021, which is a national stage application, filedunder 35 U.S.C. § 371, of PCT International Patent Application No.PCT/US2017/049158, filed on Aug. 29, 2017, and claims benefit ofpriority to U.S. Provisional Patent Application No. 62/380,871 filed onAug. 29, 2016, both of which, including their contents, are incorporatedherein by reference in their entireties.

STATEMENT OF GOVERNMENT SUPPORT

Part of the work performed during development of this invention utilizedU.S. Government funds under the National Institutes of Health/NationalEye Institute grant NIH EY 17130. The Government has certain rights inthe invention.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

This application contains a Sequence Listing file entitled052522-510D01US_ST25.txt, with a file size of about 24.75 kilobytes andcreated on or about 18 May 2021, has been submitted electronically inASCII format and is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

This invention relates generally to the field of molecular biology.Mutations in the Channelopsin variant gene (CoChop) are identified.Compositions comprising a mutant CoChop gene are used in therapeuticmethods. For example, compositions comprising a mutant CoChop geneimprove and restore vision loss.

BACKGROUND OF THE INVENTION

The retina is composed of photoreceptors (or photoreceptor cells, rodsand cones). Photoreceptors are highly specialized neurons that areresponsible for phototransduction, or the conversion of light (in theform of electromagnetic radiation) into electrical and chemical signalsthat propagate a cascade of events within the visual system, ultimatelygenerating a representation of our world.

Photoreceptor loss or degeneration severely compromises, if notcompletely inhibits, phototransduction of visual information within theretina. Loss of photoreceptor cells and/or loss of a photoreceptor cellfunction are the primary causes of diminished visual acuity, diminishedlight sensitivity, and blindness. There is a long-felt need in the artfor compositions and method that restore photosensitivity of the retinaof a subject experiencing vision loss.

SUMMARY OF THE INVENTION

The invention provides an isolated light-activated ion channelpolypeptide having the amino acid sequence of SEQ ID NO:2 and one ormore amino acid modifications. An advantage of the CoChR mutants (e.g.mutant CoChop) disclosed herein is that these mutant polypeptidesrequire less light than wild type CoChR (SEQ ID NO: 2) for activation.Thus, at the same light intensity, a greater level of ion flux and/orproton flux is observed in the mutant CoChR polypeptides than in thewild type. In some embodiments, the light-activated ion channelpolypeptide has a least one of a greater level of an ion flux and agreater level of proton flux compared to the light-activated ion channelpolypeptide of SEQ ID NO:2 when expressed in a cell membrane andcontacted with activating light (e.g. above the threshold foractivation). The light activated ion channel polypeptide has the aminoacid sequence of any of one of SEQ ID NOs: 3-10. Optionally, thepolypeptide further includes one or more amino acid modifications suchas a substitution, deletion or insertion.

In another aspect the invention provides an isolated nucleic acidmolecule that encodes for the polypeptide of the invention. Optionallythe nucleic acid sequence is operably linked to a promoter sequence.Also included in the invention are vectors containing the nucleic acidsaccording to the invention.

Also included in the invention is a cell containing the polypeptide orthe nucleic acids according to the invention. The cell is for example aphotoreceptor, a bipolar cell, a rod bipolar cell, an ON-type conebipolar cell, a retinal ganglion cell, a photosensitive retinal ganglioncell, a horizontal cell, an amacrine cell, or an AII amacrine cell. Thecell is in vitro, ex vivo or in vivo.

In other aspects the invention provides a method of changing theconductivity of a membrane by expressing in a host membrane thepolypeptide of the invention and contacting the polypeptide with a lightunder suitable conditions to change the conductivity of the hostmembrane. The host membrane is a cell membrane such as a cell membraneof a neuronal cell, a nervous system cell, a cardiac cell, a circulatorycell, a visual system cell, or an auditory system cell.

In a further aspect the invention provides methods of treating a diseaseor condition in a subject comprising administering to a subject in needthereof a therapeutically effective amount of a nucleic acid orpolypeptide according to the invention. The disease or condition is forexample, injury, brain damage, spinal cord injury, epilepsy, a metabolicdisorder, a cardiac dysfunction, vison loss, blindness, deafness,hearing loss or neurological condition.

In yet another aspect the invention features a method of improving orrestoring vision, by administering to a subject a nucleic acid orpolypeptide according to the invention. The subject is suffering from anocular disease such as macular degeneration or retinitis pigmentosa.

Improving or restoring vision includes for example increasing lightsensitivity; lowering the threshold light intensity required to elicit aphotocurrent; increasing visual evoked potential in the visual cortex;and lowering the threshold light intensity to elicit visually guidedbehavior, such as optomotor responses.

In a further aspect the invention provides methods of treating retinitispigmentosa or age related macular degeneration comprising administeringto a subject in need thereof a nucleic acid or polypeptide according tothe invention. The composition is administered by intravitreal orsubretinal injection.

Other features and advantages of the invention will be apparent from andare encompassed by the following detailed description and claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Comparison of the spectral curves of CoChR and ChR2 in HEK cellrecordings. The peak spectrum of CoChR is ˜480 nm, which is slightlymore red-shifted than that of ChR2.

FIG. 2: Sample recordings of light evoked currents of CoChRs and ChR2sin HEK cell recordings. A-D, Light-evoked currents of wt-ChR2 (A), andits three mutants, ChR2-L132C (B), ChR2-L132C/T159C (C), andChR2-L132C/T159C (D). The currents were evoked by incremental lightintensities with neutral density (ND) filters at ND=0, 2.5, 3.0, and4.0. The red traces were elicited by light at 460 nm with 2.5 neutraldensity (ND) (4.1×10¹⁵ photons/cm²s). E and F, Light-evoked currents ofwt-CoChR (E), and its mutant, CoChR-L112C (F). The red traces wereelicited by light at 480 nm with 2.5 neutral density (ND) (4.8×10¹⁵photons/cm²s).

FIG. 3: Comparison of current amplitudes for wt-CoChR and its morelight-sensitive mutants, CoChR-L112C (SEQ ID NO: 3), CoChR-T139C (SEQ IDNO: 5), CoChR-L112C/T139C (SEQ ID NO: 8), CoChR-T145A/S146A (SEQ ID NO:6), CoChR-L112C/H94E (SEQ ID NO: 9), and CoChR-L112C/H94E/K264T (SEQ IDNO: 10), in HEK cell recordings. The currents were evoked by light at480 nm with ND=2.5 (4.8×10¹⁵ photons/cm²s) and normalized to that ofwt-CoChR. Data are shown as the mean±SD.

FIG. 4: Comparison of decay time constants (off rate) for wt-CoChR andits more light-sensitive mutants, CoChR-L112C, CoChR-T139C,CoChR-L112C/T139C, CoChR-T145A/S146A, CoChR-L112C/H94E, andCoChR-L112C/H94E/K264T, in HEK cell recordings. The currents were evokedby a 10 ms light pulse at ND=0 (1.2×10¹⁸ photons/cm²s). Data are shownas the mean±SD.

FIG. 5: The relationships of the light evoked current amplitude anddecay time constant (or off rate) for wt-CoChR and its morelight-sensitive mutants, CoChR-L112C, CoChR-L112C/T139C,CoChR-L112C/H94E, and CoChR-L112C/H94E/K264T, in HEK cell recordings.

FIG. 6: Comparison of the light sensitivities of ChR2-L132C/T159C,wt-CoChR, and CoChR-L112C in retinal ganglion cells with multi-electrodearray recordings. The light intensities are shown in neutral density(ND) and photons/cm²s.

FIG. 7: Optomotor behavioral tests for the comparison of the lightsensitivity of the restored optomotor responses between ChR2-L132C/T159Sand CoChR-L112C virus vector injected mice. The relationship of spectralfrequency and threshold light intensity required to evoke optomotorresponse for ChR2-L132C/T159S and CoChR-L112C. The experiments werecarried out using a blind mouse line. Optomotor tests were conducted ina home-made optomotor assay system. The light stimulus was generated byblue LED with the wavelength of ˜470 nm. The threshold light intensityto evoke optomotor response for CoChR-L112C-expressing mice was aroundat 2-3×10¹³ photons/cm²s and for ChR2-L132C/T159S-expressing mice wasaround at 1-2×10¹⁴ photons/cm²s. Data are shown as the mean±SD.

FIG. 8: Contrast sensitivity curve for the CoChR-L112C virus vectorinjected mice based on optomotor behavioral tests. Experiments werecarried out using a blind mouse line. Optomotor tests were conducted ina virtual optomotor system (OptoMotry; CerebralMechanics, Lethbridge,AB, Canada). The illuminance inside the platform was ˜150 lux. Data areshown as the mean±SD.

FIGS. 9A-9G: Long-term stable expression of wt-CoChR and CoChR-L112C inretinal neurons mediated thought AAV vector delivery. Fluorescenceimages show the expression wt-CoChR (FIG. 9A) and its mutant CoChR-L112C(FIG. 9B) in retinal ganglion cells in C57BL/6J mice one month aftervirus injection. Fluorescence images show the expression wt-CoChR (FIG.9C) and its mutant CoChR-L112C (FIG. 9D) in retinal ganglion cells inrd1 mice six months after virus injection. Fluorescence images show theexpression CoChR-L112C in the retina of a blind mouse line viewed inwhole-mount at low (FIG. 9E) and high (FIG. 9F) magnification and inretinal vertical section (FIG. 9G) nine months after virus injection.

DETAILED DESCRIPTION

The present invention is based, in part, on the unexpected discoverythat mutations in a channelopsin variant from the green algae,Chloromonas oogama, CoChop, result in increased light sensitivity. TheCoChop mutant amino acid and nucleic acid sequences according to theinvention are referred to herein in as mCoChop. Wild-type CoChop isdescribed for example WO2015/161308, the contents of which areincorporated by reference in its entirety. The mCoChop amino acid andnucleic acid sequences according to the invention are useful in anyapplication in which a light activated ion channel is required.

In particular embodiments, the present invention features compositionand methods for the treatment of retinal degenerative diseases, such asretinitis pigmentosa or age related macular degeneration. Additionally,other diseases and disorders that are the direct result of retinaldegenerative diseases are also treated by the method of the invention.For example, advanced retinitis pigmentosa and other retinaldegenerative condition results in macular degeneration.

The channelopsin variant, CoChop was first identified via de novosequencing 127 algal transcriptomes. CoChop was identified bysynthesizing and screening for photocurrents in HEK293 cells. (See,WO2015/161308, and Klapoetke et al. Nature Methods vol. 11, No. 3 2014,the contents of each are incorporated by reference in their entireties.)

As referred to herein, “CoChop” refers to the gene that encodes achannelopsin which then forms a channelrhodopsin (CoChR) once bound toretinal. Gene constructs of the present invention refer primarily toCoChop (i.e., without the retinal), and all CoChop mutants (mCoChop)disclosed herein form functional channelrhodopsins (ChR). The methodsdisclosed herein may include delivering, mCoChop to cells with orwithout exogenous retinal. It is understood that upon expression ofmCoChop in cells (i.e., retinal neurons), endogenously available retinalbinds to the mCoChop proteins of the present invention to formfunctional light-gated channels. As such, Chop proteins, as referred toherein, can also be synonymous with ChR.

The following sequences provide non-limiting examples of wild typeCoChop mutant CoChop proteins, and polynucleotides encoding said WT andmutant Chop proteins of the invention, and forming WT and mutant ChRs ofthe invention.

Wild-type CoChR nucleic acid sequence  (SEQ ID NO: 1)ATGCTGGGAAACGGCAGCGCCATTGTGCCTATCGACCAGTGCTTTTGCCTGGCTTGGACCGACAGCCTGGGAAGCGATACAGAGCAGCTGGTGGCCAACATCCTCCAGTGGTTCGCCTTCGGCTTCAGCATCCTGATCCTGATGTTCTACGCCTACCAGACTTGGAGAGCCACTTGCGGTTGGGAGGAGGTCTACGTCTGTTGCGTCGAGCTGACCAAGGTCATCATCGAGTTCTTCCACGAGTTCGACGACCCCAGCATGCTGTACCTGGCTAACGGACACCGAGTCCAGTGGCTGAGATACGCAGAGTGGCTGCTGACTTGTCCCGTCATCCTGATCCACCTGAGCAACCTGACAGGCCTGAAGGACGACTACAGCAAGCGGACCATGAGGCTGCTGGTGTCAGACGTGGGAACCATCGTGTGGGGAGCTACAAGCGCCATGAGCACAGGCTACGTCAAGGTCATCTTCTTCGTGCTGGGTTGCATCTACGGCGCCAACACCTTCTTCCACGCCGCCAAGGTGTATATCGAGAGCTACCACGTGGTGCCAAAGGGCAGACCTAGAACCGTCGTGCGGATCATGGCTTGGCTGTTCTTCCTGTCTTGGGGCATGTTCCCCGTGCTGTTCGTCGTGGGACCAGAAGGATTCGACGCCATCAGCGTGTACGGCTCTACCATTGGCCACACCATCATCGACCTCATGAGCAAGAATTGTTGGGGCCTGCTGGGACACTATCTGAGAGTGCTGATCCACCAGCACATCATCATCTACGGCGACATCCGCAAGAAGACCAAGATCAACGTGGCCGGCGAGGAGATGGAAGTGGAGACCATGGTGGACCAGGAGGACGAGGAGACAGTGWild-type CoChR amino acid sequence  (SEQ ID NO: 2) MLGNGSAIVPIDQCFCLAWTDSLGSDTEQLVANILQWFAFGFSILILMFYAYQTWRATCGWEEVYVCCVELTKVIIEFFHEFDDPSMLYLANGHRVQWLRYAEWLLTCPVILIHLSNLTGLKDDYSKRTMRLLVSDVGTIVWGATSAMSTGYVKVIFFVLGCIYGANTFFHAAKVYIESYHVVPKGRPRTVVRIMAWLFFLSWGMFPVLFVVGPEGFDAISVYGSTIGHTIIDLMSKNCWGLLGHYLRVLIHQHIIIYGDIRKKTKINVAGEEMEVETMVDQEDEETV Mutant CoChR amino acid sequence L112C (SEQ ID NO: 3)MLGNGSAIVPIDQCFCLAWTDSLGSDTEQLVANILQWFAFGFSILILMFYAYQTWRATCGWEEVYVCCVELTKVIIEFFHEFDDPSMLYLANGHRVQWLRYAEWLLTCPVICIHLSNLTGLKDDYSKRTMRLLVSDVGTIVWGATSAMSTGYVKVIFFVLGCIYGANTFFHAAKVYIESYHVVPKGRPRTVVRIMAWLFFLSWGMFPVLFVVGPEGFDAISVYGSTIGHTIIDLMSKNCWGLLGHYLRVLIHQHIIIYGDIRKKTKINVAGEEMEVETMVDQEDEETV Mutant CoChR amino acid sequence C68S/V69I (SEQ ID NO: 4)MLGNGSAIVPIDQCFCLAWTDSLGSDTEQLVANILQWFAFGFSILILMFYAYQTWRATCGWEEVYVCSIELTKVIIEFFHEFDDPSMLYLANGHRVQWLRYAEWLLTCPVILIHLSNLTGLKDDYSKRTMRLLVSDVGTIVWGATSAMSTGYVKVIFFVLGCIYGANTFFHAAKVYIESYHVVPKGRPRTVVRIMAWLFFLSWGMFPVLFVVGPEGFDAISVYGSTIGHTIIDLMSKNCWGLLGHYLRVLIHQHIIIYGDIRKKTKINVAGEEMEVETMVDQEDEETV Mutant CoChR amino acid sequence T139C (SEQ ID NO: 5)MLGNGSAIVPIDQCFCLAWTDSLGSDTEQLVANILQWFAFGFSILILMFYAYQTWRATCGWEEVYVCCVELTKVIIEFFHEFDDPSMLYLANGHRVQWLRYAEWLLTCPVILIHLSNLTGLKDDYSKRTMRLLVSDVGCIVWGATSAMSTGYVKVIFFVLGCIYGANTFFHAAKVYIESYHVVPKGRPRTVVRIMAWLFFLSWGMFPVLFVVGPEGFDAISVYGSTIGHTIIDLMSKNCWGLLGHYLRVLIHQHIIIYGDIRKKTKINVAGEEMEVETMVDQEDEETV Mutant CoChR amino acid sequence T145A/S146A (SEQ ID NO: 6)MLGNGSAIVPIDQCFCLAWTDSLGSDTEQLVANILQWFAFGFSILILMFYAYQTWRATCGWEEVYVCCVELTKVIIEFFHEFDDPSMLYLANGHRVQWLRYAEWLLTCPVILIHLSNLTGLKDDYSKRTMRLLVSDVGTIVWGAAAAMSTGYVKVIFFVLGCIYGANTFFHAAKVYIESYHVVPKGRPRTVVRIMAWLFFLSWGMFPVLFVVGPEGFDAISVYGSTIGHTIIDLMSKNCWGLLGHYLRVLIHQHIIIYGDIRKKTKINVAGEEMEVETMVDQEDEETV Mutant CoChR amino acid sequence C68T/V69L (SEQ ID NO: 7)MLGNGSAIVPIDQCFCLAWTDSLGSDTEQLVANILQWFAFGFSILILMFYAYQTWRATCGWEEVYVCTLELTKVIIEFFHEFDDPSMLYLANGHRVQWLRYAEWLLTCPVILIHLSNLTGLKDDYSKRTMRLLVSDVGTIVWGATSAMSTGYVKVIFFVLGCIYGANTFFHAAKVYIESYHVVPKGRPRTVVRIMAWLFFLSWGMFPVLFVVGPEGFDAISVYGSTIGHTIIDLMSKNCWGLLGHYLRVLIHQHIIIYGDIRKKTKINVAGEEMEVETMVDQEDEETV Mutant CoChR amino acid sequence L112C/T139C (SEQ ID NO: 8)MLGNGSAIVPIDQCFCLAWTDSLGSDTEQLVANILQWFAFGFSILILMFYAYQTWRATCGW LEVYVCCVELTKVIIEFFHEFDDPSMLYLANGHRVQWLRYAEWLLTCPVICIHLSNLTGLKD DYSKRTMRLLVSDVGCIVWGATSAMSTGYVKVIFFVLGCIYGANTFFHAAKVYIESYHVVP KGRPRTVVRIMAWLFFLSWGMFPVLFVVGPEGFDAISVYGSTIGHTIIDLMSKNCWGLLGH YLRVLIHQHIIIYGDIRKKTKINVAGEEMEVETMVDQEDEETV Mutant CoChR amino acid sequence L112C/H94E  (SEQ ID NO: 9)MLGNGSAIVPIDQCFCLAWTDSLGSDTEQLVANILQWFAFGFSILILMFYAYQTWRATCGWLEVYVCCVELTKVIIEFFHEFDDPSMLYLANGERVQWLRYAEWLLTCPVICIHLSNLTGLKDDYSKRTMRLLVSDVGTIVWGATSAMSTGYVKVIFFVLGCIYGANTFFHAAKVYIESYHVVPKGRPRTVVRIMAWLFFLSWGMFPVLFVVGPEGFDAISVYGSTIGHTIIDLMSKNCWGLLGHYLRVLIHQHIIIYGDIRKKTKINVAGEEMEVETMVDQEDEETV Mutant CoChR amino acid sequence L112C/H94E/K264T  (SEQ ID NO: 10)MLGNGSAIVPIDQCFCLAWTDSLGSDTEQLVANILQWFAFGFSILILMFYAYQTWRATCGWLEVYVCCVELTKVIIEFFHEFDDPSMLYLANGERVQWLRYAEWLLTCPVICIHLSNLTGLKDDYSKRTMRLLVSDVGTIVWGATSAMSTGYVKVIFFVLGCIYGANTFFHAAKVYIESYHVVPKGRPRTVVRIMAWLFFLSWGMFPVLFVVGPEGFDAISVYGSTIGHTIIDLMSKNCWGLLGHYLRVLIHQHIIIYGDIRKTTKINVAGEEMEVETMVDQEDEETV

The present invention also encompasses CoChop proteins and nucleic acidsthat encode a biologically active fragment or a conservative amino acidsubstitution or other mutation variant of CoChop. Smaller fragments ofwild-type CoChop, wherein at least one amino acid is mutated orconservatively substituted may also be useful in the present invention.In other embodiments, the CoChop polypeptides and nucleic acids of thepresent invention can be up to, or about, 275 amino acids long, 250amino acids long, 225 amino acids long, 200 amino acids long, 175 aminoacids long, or 160 amino acids long.

In some embodiments, the disclosure provides derivatives, variants, ormutants of one or more CoChop polypeptides disclosed herein. In someembodiments, the derivative, variant, or mutant contains one or moreamino acid substitutions compared to the amino acid sequence of thenative polypeptide (e.g. SEQ ID NO: 2). In some embodiments, one to 20amino acids are substituted. In some embodiments, the derivative,variant, or mutant contains about 1, about 2, about 3, about 4, about 5,about 6, about 7, about 8, about 9, or about 10 amino acid substitutionscompared to the amino acid sequence of the native therapeutic peptideagent. In some embodiments, the derivative, variant, or mutant containsone or more amino acid deletions compared to the amino acid sequence ofthe native polypeptide (e.g. SEQ ID NO: 2). In some embodiments, one to20 amino acids are deleted compared to the amino acid sequence of thenative polypeptide (e.g. SEQ ID NO: 2). In some embodiments, thederivative, variant, or mutant has about 1, about 2, about 3, about 4,about 5, about 6, about 7, about 8, about 9, or about 10 amino aciddeletions compared to the amino acid sequence of the native polypeptide(e.g. SEQ ID NO: 2). In some embodiments, one to ten amino acids aredeleted at either terminus compared to the amino acid sequence of thenative polypeptide (e.g. SEQ ID NO: 2). In some embodiments, one to tenamino acids are deleted from both termini compared to the amino acidsequence of the native polypeptide (e.g. SEQ ID NO: 2). In someembodiments, the amino acid sequence of the derivative, variant, ormutant is at least about 70% identical to the amino acid sequence of thenative polypeptide (e.g. SEQ ID NO: 2). In some embodiments, the aminoacid sequence of the derivative, variant, or mutant is about 70%, about80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%,or about 99% identical to the amino acid sequence of the nativepolypeptide (e.g. SEQ ID NO: 2).

Mutant CoChop proteins of the invention also demonstrate slower channelkinetics. Higher light sensitivity was found to correlate with slowerchannel kinetics, indicating a trade-off between light sensitivity andchannel kinetics. mCoChop proteins that form the ChR proteins of thepresent invention may also comprise additional mutations ormodifications that may improve channel kinetics, or increase thedeactivation rate. Particularly preferred CoChop mutants balance thethreshold of light sensitivity with channel kinetics.

For example, mutant ChR proteins of the invention achieve greater lightsensitivity through the prolongation of the channel open state.Consequently, each mutant ChR channel conducts a greater photocurrentthan a wild type ChR channel when activated by the same lightintensities. Therefore, the mutant channels are activated by lightintensities that are lower than those required for activation of thewild type ChR channels. Quantitatively, detectable spiking activity ofretinal ganglion cells expressing mutant ChR proteins can be elicited bya light intensity that is 1.5-2 log units lower than the light intensityrequired to elicit spiking activity from retinal ganglion cellsexpressing wild type ChR. Thus, the light intensities required toactivate the mutant ChR proteins are close to or fall within the rangeof normal outdoor lighting conditions.

Nucleic Acids, Vectors and Recombinant Viruses

In some aspect of the invention, the compositions and methods of thedisclosure provide for the delivery of a nucleic acid encoding mCoChop(mutant CoChop) to cells in a subject or patient in need thereof. Insome cases, delivery of the nucleic acid may be referred to as genetherapy.

The composition and methods of the disclosure provide for any suitablemethod for delivery of the mCoChop nucleic acid. In some cases, deliveryof the nucleic acid may be performed using any suitable “vector”(sometimes also referred to as “gene delivery” or “gene transfer”vehicle). Vector, delivery vehicle, gene delivery vehicle or genetransfer vehicle, may refer to any suitable macromolecule or complex ofmolecules comprising a polynucleotide to be delivered to a target cell.In some cases, a target cell may be any cell to which the nucleic acidor gene is delivered. The polynucleotide to be delivered may comprise acoding sequence of interest in gene therapy, such as the mCoChop gene.

For example, suitable vectors may include but are not limited to, viralvectors such as adenoviruses, adeno-associated viruses (AAV), andretroviruses, liposomes, other lipid-containing complexes, and othermacromolecular complexes capable of mediating delivery of apolynucleotide to a target cell.

In some cases, a vector may be an organic or inorganic molecule. In somecases, a vector may be small molecule (i.e. <5 kD), or a macromolecule(i.e. >5 kD). For example a vector may include but is not limited toinert, non-biologically active molecules such as metal particles. Insome cases, a vector may be gold particles.

In some aspects, a vector may comprise a recombinant viral vector thatincorporates one or more nucleic acids. As described herein, nucleicacids may refer to polynucleotides. Nucleic acid and polynucleotide maybe used interchangeably. In some cases nucleic acids may comprise DNA orRNA. In some aspects, nucleic acids may include DNA or RNA for theexpression of mCoChop. In some aspects RNA nucleic acids may include butare not limited to a transcript of a gene of interest (e.g. mCoChop),introns, untranslated regions, termination sequences and the like. Inother cases, DNA nucleic acids may include but are not limited tosequences such as hybrid promoter gene sequences, strong constitutivepromoter sequences, the gene of interest (e.g. mCoChop), untranslatedregions, termination sequences and the like. In some cases, acombination of DNA and RNA may be used.

As described in the disclosure herein, the term “expression construct”is meant to include any type of genetic construct containing a nucleicacid or polynucleotide coding for gene products in which part or all ofthe nucleic acid encoding sequence is capable of being transcribed. Thetranscript may be translated into a protein. In some aspects it may bepartially translated or not translated. In certain aspects, expressionincludes both transcription of a gene and translation of mRNA into agene product. In other aspects, expression only includes transcriptionof the nucleic acid encoding genes of interest.

In one aspect, the present disclosure provides a recombinant virus, suchas adeno-associated virus (rAAV) as a vector to mediate the expressionof mCoChop.

In some cases, the viral vector of the disclosure may be measured as pfu(plaque forming units). In some cases, the pfu of recombinant virus, orviral vector of the compositions and methods of the disclosure may beabout 10⁸ to about 5×10¹⁰ pfu. In some cases, recombinant viruses ofthis disclosure are at least about 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸,6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹,7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, and 5×10¹⁰ pfu. Insome cases, recombinant viruses of this disclosure are at most about1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹,2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰,3×10¹⁰, 4×10¹⁰, and 5×10¹⁰ pfu.

In some cases, the viral vector of the disclosure may be measured asvector genomes. In some cases, recombinant viruses of this disclosureare 1×10¹⁰ to 3×10¹² vector genomes. In some cases, recombinant virusesof this disclosure are 1×10⁹ to 3×10¹³ vector genomes. In some cases,recombinant viruses of this disclosure are 1×10⁸ to 3×10¹⁴ vectorgenomes. In some cases, recombinant viruses of the disclosure are atleast about 1×10¹, 1×10², 1×10³, 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸,1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², 1×10¹³, 1×10¹⁴′ 1×10¹⁵, 1×10¹⁶, 1×10¹⁷,and 1×10¹⁸ vector genomes.

In some cases, the viral vector of the disclosure may be measured usingmultiplicity of infection (MOI). In some cases, MOI may refer to theratio, or multiple of vector or viral genomes to the cells to which thenucleic may be delivered. In some cases, the MOI may be 1×10⁶. In somecases, the MOI may be 1×10⁵-1×10⁷. In some cases, the MOI may be1×10⁴-1×10⁸. In some cases, recombinant viruses of the disclosure are atleast about 1×10¹, 1×10², 1×10³, 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸,1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², 1×10¹³, 1×10¹⁴, 1×10¹⁵, 1×10¹⁶, 1×10¹⁷,and 1×10¹⁸ MOI. In some cases, recombinant viruses of this disclosureare 1×10⁸ to 3×10¹⁴ MOI.

In some aspects the nucleic acid may be delivered without the use of avirus (i.e. with a non-viral vector), and may be measured as thequantity of nucleic acid. Generally, any suitable amount of nucleic acidmay be used with the compositions and methods of this disclosure. Insome cases, nucleic acid may be at least about 1 pg, 10 pg, 100 pg, 1pg, 10 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800pg, 900 pg, 1 μg, 10 μg, 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg,700 μg, 800 μg, 900 μg, 1 ng, 10 ng, 100 ng, 200 ng, 300 ng, 400 ng, 500ng, 600 ng, 700 ng, 800 ng, 900 ng, 1 mg, 10 mg, 100 mg, 200 mg, 300 mg,400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg 1 g, 2 g, 3 g, 4 g, or 5g. In some cases, nucleic acid may be at most about 1 pg, 10 pg, 100 pg,1 pg, 10 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800pg, 900 pg, 1 μg, 10 μg, 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg,700 μg, 800 μg, 900 μg, 1 ng, 10 ng, 100 ng, 200 ng, 300 ng, 400 ng, 500ng, 600 ng, 700 ng, 800 ng, 900 ng, 1 mg, 10 mg, 100 mg, 200 mg, 300 mg,400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1 g, 2 g, 3 g, 4 g, or 5g. In some aspects, a self-complementary vector (sc) may be used. Theuse of self-complementary AAV vectors may bypass the requirement forviral second-strand DNA synthesis and may lead to greater rate ofexpression of the transgene protein, as provided by Wu, Hum Gene Ther.2007, 18(2):171-82, incorporated by reference herein.

The compositions and methods of the disclosure provide for any suitableviral nucleic acid delivery systems including but not limited to use ofat least one of an adeno-associated virus (AAV), adenovirus,helper-dependent adenovirus, retrovirus, herpes simplex virus,lentivirus, poxvirus, hemagglutination virus of Japan-liposome (HVJ)complex, Moloney murine leukemia virus, and HIV-based virus. Preferably,the viral vector comprises a strong eukaryotic promoter operably linkedto the polynucleotide.

Generally, any suitable viral vectors may be engineered to be optimizedfor use with the compositions and methods of the disclosure. Forexample, viral vectors derived from adenovirus (Ad) or adeno-associatedvirus (AAV) may be used. Both human and non-human viral vectors can beused and the recombinant viral vector can be altered such that it may bereplication-defective in humans. Where the vector is an adenovirus, thevector can comprise a polynucleotide having a promoter operably linkedto a gene encoding the mCoChop protein and is replication-defective inhumans.

To combine advantageous properties of two viral vector systems, hybridviral vectors may be used to deliver a nucleic acid encoding a mCoChopprotein to a target cell or tissue. Standard techniques for theconstruction of hybrid vectors are well-known to those skilled in theart. Such techniques can be found, for example, in Sambrook, et al., InMolecular Cloning: A laboratory manual. Cold Spring Harbor, N.Y. or anynumber of laboratory manuals that discuss recombinant DNA technology.Double-stranded AAV genomes in adenoviral capsids containing acombination of AAV and adenoviral ITRs may be used to transduce cells.In another variation, an AAV vector may be placed into a “gutless”,“helper-dependent” or “high-capacity” adenoviral vector. Adenovirus/AAVhybrid vectors are discussed in Lieber et al., J. Virol. 73:9314-9324,1999. Retrovirus/adenovirus hybrid vectors are discussed in Zheng etal., Nature Biotechnol. 18:176-186, 2000.

Retroviral genomes contained within an adenovirus may integrate withinthe target cell genome and effect stable gene expression.

Replication-defective recombinant adenoviral vectors can be produced inaccordance with known techniques. See, Quantin, et al., Proc. Natl.Acad. Sci. USA, 89:2581-2584 (1992); Stratford-Perricadet, et al., J.Clin. Invest., 90:626-630 (1992); and Rosenfeld, et al., Cell,68:143-155 (1992).

Additionally preferred vectors may include but are not limited to viralvectors, fusion proteins and chemical conjugates. Retroviral vectorsinclude Moloney murine leukemia viruses and HIV-based viruses. In somecases a HIV-based viral vector may be used, wherein the HIV-based viralvector comprises at least two vectors wherein the gag and pol genes arefrom an HIV genome and the env gene is from another virus. DNA viralvectors may be used. These vectors include pox vectors such as orthopoxor avipox vectors, herpesvirus vectors such as a herpes simplex I virus(HSV) vector [Geller, A. I. et al., J. Neurochem, 64: 487 (1995); Lim,F., et al., in DNA Cloning: Mammalian Systems, D. Glover, Ed. (OxfordUniv. Press, Oxford England) (1995); Geller, A. I. et al., Proc Natl.Acad. Sci.: U.S.A.: 90 7603 (1993); Geller, A. I., et al., Proc Natl.Acad. Sci. USA: 87:1149 (1990)], Adenovirus Vectors [LeGal LaSalle etal., Science, 259:988 (1993); Davidson, et al., Nat. Genet. 3: 219(1993); Yang, et al., J. Virol. 69: 2004 (1995)] and Adeno-associatedVirus Vectors [Kaplitt, M. G., et al., Nat. Genet. 8:148 (1994)],incorporated by reference herein.

Other viral vectors that can be used in accordance with the presentdisclosure include herpes simplex virus (HSV)-based vectors. HSV vectorsdeleted of one or more immediate early genes (IE) are advantageousbecause they are generally non-cytotoxic, persist in a state similar tolatency in the target cell, and afford efficient target celltransduction. Recombinant HSV vectors can incorporate approximately 30kb of heterologous nucleic acid.

Retroviruses, such as C-type retroviruses and lentiviruses, may also beused in the disclosure. For example, retroviral vectors may be based onmurine leukemia virus (MLV)., as provided by Hu and Pathak, Pharmacol.Rev. 52:493511, 2000 and Fong et al., Crit. Rev. Ther. Drug CarrierSyst. 17:1-60, 2000, incorporated by reference herein. MLV-based vectorsmay contain up to 8 kb of heterologous (therapeutic) DNA in place of theviral genes. Additional retroviral vectors may be used including but notlimited to replication-defective lentivirus-based vectors, includinghuman immunodeficiency (HIV)-based vectors, as provided by Vigna andNaldini, J. Gene Med. 5:308-316, 2000 and Miyoshi et al., J. Virol.72:8150-8157, 1998, incorporated by reference herein. Lentiviral vectorsmay be advantageous in that they are capable of infecting both activelydividing and non-dividing cells. They may also be highly efficient attransducing human epithelial cells.

Lentiviral vectors for use in the disclosure may be derived from humanand non-human (including SIV) lentiviruses. Examples of lentiviralvectors include nucleic acid sequences required for vector propagationas well as a tissue-specific promoter operably linked to a mCoChop gene.Nucleic acid sequences may include the viral LTRs, a primer bindingsite, a polypurine tract, att sites, and an encapsidation site.

A lentiviral vector may be packaged into any suitable lentiviral capsid.The substitution of one particle protein with another from a differentvirus is referred to as “pseudotyping”. The vector capsid may containviral envelope proteins from other viruses, including murine leukemiavirus (MLV) or vesicular stomatitis virus (VSV). The use of the VSVG-protein yields a high vector titer and results in greater stability ofthe vector virus particles.

Alphavirus-based vectors, such as those made from semliki forest virus(SFV) and sindbis virus (SIN), may also be used in the disclosure. Useof alphaviruses is described in Lundstrom, K., Intervirology 43:247-257,2000 and Perri et al., Journal of Virology 74:9802-9807, 2000,incorporated by reference herein.

Recombinant, replication-defective alphavirus vectors may beadvantageous because they are capable of high-level heterologous(therapeutic) gene expression, and can infect a wide target cell range.Alphavirus replicons may be targeted to specific cell types bydisplaying on their virion surface a functional heterologous ligand orbinding domain that would allow selective binding to target cellsexpressing a cognate binding partner. Alphavirus replicons may establishlatency, and therefore long-term heterologous nucleic acid expression ina target cell. The replicons may also exhibit transient heterologousnucleic acid expression in the target cell.

Pox viral vectors may introduce a gene into the cell's cytoplasm. Avipoxvirus vectors may result in only a short-term expression of the gene ornucleic acid. Adenovirus vectors, adeno-associated virus vectors andherpes simplex virus (HSV) vectors may be used with the compositions andmethods of the disclosure. The adenovirus vector may result in ashorter-term expression (e.g., less than about a month) thanadeno-associated virus, in some aspects, and may exhibit much longerexpression. The particular vector chosen may depend upon the target celland the condition being treated.

Adeno-associated viruses (AAV) are small non-enveloped single-strandedDNA viruses. They are non-pathogenic human parvoviruses and may bedependent on helper viruses, including adenovirus, herpes simplex virus,vaccinia virus and CMV, for replication. Exposure to wild-type (wt) AAVis not associated or known to cause any human pathologies and is commonin the general population, usually occurring in the first decade of lifein association with an adenoviral infection.

As described herein, “AAV” refers to Adeno-associated virus “rAAV”refers to a recombinant adeno-associated virus.

In some cases, the wild-type AAV encodes rep and cap genes. The rep geneis required for viral replication and the cap gene is required forsynthesis of capsid proteins. Through a combination of alternativetranslation start and splicing sites, the small genome may be able toexpress four rep and three cap gene products. The rep gene products andsequences in the inverted terminal repeats (145 bp ITRs, which flank thegenome) may be critical in this process. To date, 11 serotypes of AAVhave been isolated. The compositions and methods of the disclosureprovide for use of any suitable AAV serotype. In some aspects, the AAVis selected from the group consisting of: AAV1, AAV2, AAV2.5, AAV3,AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, rh10, andhybrids thereof. AAV2 may be used with composition and methods of thedisclosure.

AAV2 is the most characterized. rAAV2 has been shown to be able tomediate long-term transgene expression in the eyes of many species ofanimals. In rats, rAAV mediated reporter gene (green fluorescentprotein) was still present at 18 months post injection. In monkeys, thesame reporter gene was present at 17 months post injection.

Vectors can comprise components or functionalities that further modulategene delivery and/or gene expression, or that otherwise providebeneficial properties to the targeted cells. Such other componentsinclude, for example, components that influence binding or targeting tocells (including components that mediate cell-type or tissue-specificbinding); components that influence uptake of the vector nucleic acid bythe cell; components that influence localization of the polynucleotidewithin the cell after uptake (such as agents mediating nuclearlocalization); and components that influence expression of thepolynucleotide. Such components also might include markers, such asdetectable and/or selectable markers that can be used to detect orselect for cells that have taken up and are expressing the nucleic aciddelivered by the vector. Such components can be provided as a naturalfeature of the vector (such as the use of certain viral vectors whichhave components or functionalities mediating binding and uptake), orvectors can be modified to provide such functionalities.

Selectable markers can be positive, negative or bifunctional. Positiveselectable markers allow selection for cells carrying the marker,whereas negative selectable markers allow cells carrying the marker tobe selectively eliminated. A variety of such marker genes have beendescribed, including bifunctional (i.e., positive/negative) markers(see, e.g., Lupton, S., WO 92/08796, published May 29, 1992; and Lupton,S., WO 94/28143, published Dec. 8, 1994). Examples of negativeselectable markers may include the inclusion of resistance genes toantibiotics, such as ampicillin or kanamycin. Such marker genes canprovide an added measure of control that can be advantageous in genetherapy contexts. A large variety of such vectors are known in the artand are generally available.

In many of the viral vectors compatible with methods of the disclosure,one or more promoters can be included in the vector to allow more thanone heterologous gene to be expressed by the vector. Further, the vectorcan comprise a sequence which encodes a signal peptide or other moietywhich facilitates expression of the mCoChop protein from the targetcell.

The nucleic acid encoding a gene product may be under transcriptionalcontrol by a promoter. A “promoter”, as provided herein, refers to asuitable DNA sequence required to initiate transcription of a gene. Thephrase “under transcriptional control” means that the promoter is in thecorrect location and orientation in relation to the nucleic acid tocontrol RNA polymerase initiation and expression of the gene. In somecases, promoter may include a “strong” or constitutively activepromoter. For example, the CMV promoter may be used as known in the arta constitutively active promoter. In some cases, the CMV promoter maycomprise additional regulatory elements for promoting expression.

In some cases a promoter may refer to a “weak” promoter, or sequencethat yields lower levels of mCoChop protein than a strong promoter. Insome cases a promoter may be used such that the promoter drivesselective expression of mCoChop. In some cases a promoter or otherregulatory elements used in combination with other sequences asdescribed herein may be used to drive selective expression of mCoChop inan eye cell, or eye tissue.

Additionally, “promoter”, may also be used herein interchangeably torefer to any additional suitable transcriptional control modules thatmay be present around the initiation site for RNA polymerases. Thecompositions and methods of this disclosure may use any suitablepromoters and transcriptional control modules for expression of atransgene. Additional transcriptional control modules may include butare not limited to elements such as HSV thymidine kinase (tk) and SV40early transcription units. Generally, promoters may be composed ofdiscrete functional modules, each consisting of approximately 7-20 bp ofDNA, or 20-5000 bp of DNA, and contain one or more recognition sites fortranscriptional activator or repressor proteins. The composition andmethods of the disclosure provide for any suitable regulatory sequencesor combination thereof. In some cases, these transcriptional controlmodule sequences may be referred to or identified as enhancer orrepressor sequences.

At least one module in each promoter functions to position the startsite for RNA synthesis. One example is the TATA box. Other examples mayinclude some promoters that lack a TATA box, such as the promoter forthe mammalian terminal deoxynucleotidyl transferase gene and thepromoter for the SV40 late genes, a discrete element overlying the startsite itself helps to fix the place of initiation.

Additional promoter elements regulate the frequency of transcriptionalinitiation. Generally, these are located in a region 30-110 bp upstreamof the start site, although a number of promoters may contain functionalelements downstream of the start site as well. The spacing betweenpromoter elements frequently may be flexible, so that promoter functionis preserved when elements are inverted or moved relative to oneanother. In the tk promoter for example, the spacing between promoterelements can be increased to 50 bp apart before activity begins todecline. Depending on the promoter, individual elements may position tofunction either co-operatively or independently to activatetranscription.

The compositions and methods of the disclosure provide for any suitablesequences for the control of expression of a nucleic acid sequence ofinterest in the targeted cell. Thus, where a human cell is targeted, thenucleic acid coding region may be engineered to be adjacent to and underthe control of a promoter that is capable of being expressed in a humancell. Generally, such a promoter might include either a human or viralpromoter.

In various aspects of the disclosure, the human cytomegalovirus (CMV)immediate early (IE) enhancer, a chicken β-actin promoter, a chickenβ-actin exon 1, a hybrid chicken β-actin and rabbit β-globin intron, asimian virus 40 polyadenylation signal can be used to obtain a highlevel of expression of the coding sequence of interest (e.g. mCoChop).

The use of other viral or mammalian cellular or bacterial phagepromoters which are well-known in the art to achieve expression of acoding sequence of interest is contemplated as well, provided that thelevels of expression are sufficient for a given purpose. In someaspects, prokaryotic regulatory sequences may be present in the vector,such as the T7 RNA polymerase promoter sequence. In other aspects, thevector is free from such regulatory sequences. By employing a promoterwith known properties, the level and pattern of expression of theprotein of interest following transfection or transformation can beoptimized.

Selection of a promoter that is regulated in response to specificphysiologic or synthetic signals can permit inducible expression of thegene product. For example in the case where expression of a transgene,or transgenes when a multicistronic vector is utilized, is toxic to thecells in which the vector is produced in, it may be desirable toprohibit or reduce expression of one or more of the transgenes. Examplesof transgenes that may be toxic to the producer cell line arepro-apoptotic and cytokine genes. Several inducible promoter systems areavailable for production of viral vectors where the transgene productmay be toxic. The composition and methods of the disclosure provide forany suitable combination of promoter sequence, regulatory sequences andtransgene. In some cases, a combination of sequences may result in notoxicity to the cell. In some cases, a combination of sequences mayresult in high toxicity to the cell. In some cases, a combination ofsequences may result in moderate levels of toxicity in the cell.

In some circumstances, it may be desirable to regulate expression of atransgene in a gene therapy vector. For example, different viralpromoters with varying strengths of activity may be utilized dependingon the level of expression desired. In mammalian cells, the CMVimmediate early promoter may be used to provide strong transcriptionalactivation. Modified versions of the CMV promoter that are less potenthave also been used when reduced levels of expression of the transgeneare desired. When expression of a transgene in hematopoietic cells isdesired, retroviral promoters such as the LTRs (Long Terminal Repeat)from MLV or MMTV are often used. Other viral promoters that may be useddepending on the desired effect include SV40, RSV LTR, HIV-1 and HIV-2LTR, adenovirus promoters such as from the E1A, E2A, or MLP region, AAVLTR, cauliflower mosaic Virus, HSV-TK, and avian sarcoma virus.

In some cases, promoters or regulatory sequence elements may be used todirect selective expression in eye cells or eye tissue. For example,promoter, sequence elements or regulatory sequences found in specificeye cell types, such as retinal pigment epithelial cells, may be used ina suitable expression construct (e.g., the RPE65 or VMD2 promoter).

The selection of appropriate promoters can be readily accomplished. Insome cases a high expression, or strong promoter may be used.

Other elements that can enhance expression can also be included such asan enhancer or a system that results in high levels of expression suchas a tat gene and tar element. This cassette can then be inserted into avector, e.g., a plasmid vector such as, pUC19, pUC118, pBR322, or otherknown plasmid vectors, that includes, for example, an E. coli origin ofreplication. See, Sambrook, et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory press, (1989). Promoters arediscussed infra. The plasmid vector may also include a selectable markersuch as the .beta.-lactamase gene for ampicillin resistance, providedthat the marker polypeptide does not adversely affect the metabolism ofthe organism being treated. The cassette can also be bound to a nucleicacid binding moiety in a synthetic delivery system, such as the systemdisclosed in WO 95/22618, incorporated by reference herein. Generallypromoter sequences and/or any associated regulatory sequences maycomprise about at least 150 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp,700 bp, 800 bp, 900 bp, 1000 bp, 2000 bp, 3000 bp, 4000 bp, 5000 bp or10000 bp. Promoter sequences and any associated regulatory sequences,may comprise about at most 150 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600bp, 700 bp, 800 bp, 900 bp, 1000 bp, 2000 bp, 3000 bp, 4000 bp, 5000 bpor 10000 bp.

In some aspects, the recombinant virus or plasmid comprises a promoterselected from cytomegalovirus (CMV) promoter, Rous sarcoma virus (RSV)promoter, and MMT promoter, EF-1 alpha promoter, UB6 promoter, chickenbeta-actin promoter, CAG promoter, RPE65 promoter and opsin promoter.

In some aspects, an antibiotic marker is used in the process forproduction of the recombinant virus. Antibiotic resistance markers maybe used to identify positive transgenic cells in the generation ofrecombinant virus. For example markers conferring resistance may includebut are not limited to kanamycin, gentamicin, ampicillin,chloramphenicol, tetracycline, doxycycline, or hygromycin. In someaspects, the antibiotic resistance gene is a non-beta-lactam antibioticresistance gene such as kanamycin.

In some aspects, the recombinant virus and/or plasmid used to generaterecombinant virus, comprise a sequence encoding a replication originsequence, such as those provided herein. Origin of replicationsequences, generally provide sequence useful for propagating a plasmid.

In some aspects, the recombinant virus and/or plasmid used to generaterecombinant virus, comprise an enhancer, such as those provided herein.Preferably the enhancer is a CMV immediate early enhancer.

In some aspects, the recombinant virus and/or plasmid used to generaterecombinant virus, comprise a poly A (polyadenylation) sequence, such asthose provided herein (e.g. SV40 poly A sequence.). Generally, anysuitable polyA sequence may be used for the desired expression of thetransgene (i.e. mCoChop). For example, in some cases, the presentdisclosure provides for a sequence comprising SV40 polyA sequence, orportion of SV40 polyA sequence. In some cases, the present disclosureprovides for polyA sequences comprising a combination of one or morepolyA sequences or sequence elements. In some cases, no polyA sequenceis used. In some cases one or more polyA sequences may be referred to asuntranslated regions (UTRs), 3′ UTRs, or termination sequences.Preferably, a SV40 polyA sequence is used.

A polyA sequence may comprise a length of 1-10 bp, 10-20 bp, 20-50 bp,50-100 bp, 100-500 bp, 500 bp-1 Kb, 1 Kb-2 Kb, 2 Kb-3 Kb, 3 Kb-4 Kb, 4Kb-5 Kb, 5 Kb-6 Kb, 6 Kb-7 Kb, 7 Kb-8 Kb, 8 Kb-9 Kb, and 9 Kb-10 Kb inlength. A polyA sequence may comprise a length of at least 1 bp, 2 bp, 3bp, 4 bp, 5 bp, 6 bp, 7 bp, 8 bp, 9 bp, 10 bp, 20 bp, 30 bp, 40 bp, 50bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500 bp,600 bp, 700 bp, 800 bp, 900 bp, 1 Kb, 2 Kb, 3 Kb, 4 Kb, 5 Kb, 6 Kb, 7Kb, 8 Kb, 9 Kb, and 10 Kb in length. A polyA sequence may comprise alength of at most 1 bp, 2 bp, 3 bp, 4 bp, 5 bp, 6 bp, 7 bp, 8 bp, 9 bp,10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp,200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1 Kb, 2Kb, 3 Kb, 4 Kb, 5 Kb, 6 Kb, 7 Kb, 8 Kb, 9 Kb, and 10 Kb in length.

In some cases, polyA sequences may be optimized for various parametersaffecting protein expression, including but not limited to mRNAhalf-life of the transgene in the cell, stability of the mRNA of thetransgene or transcriptional regulation. For example, polyA sequencesmaybe altered to increase mRNA transcript of the transgene, which mayresult in increased protein expression. In some cases, the polyAsequences maybe altered to decrease the half-life of the mRNA transcriptof the transgene, which may result in decreased protein expression.

In certain aspects of the disclosure, the use of internal ribosome entrysite (IRES) or foot-mouth disease virus (FMDV) elements may be used tocreate multigene, or polycistronic, messages. IRES elements are able tobypass the ribosome scanning model of 5′ methylated Cap dependenttranslation and begin translation at internal sites. IRES elements fromtwo members of the picornavirus family (poliovirus andencephalomyocarditis) have been described, as well an IRES from amammalian message. IRES elements can be linked to heterologous openreading frames. Multiple open reading frames can be transcribedtogether, each separated by an IRES, creating polycistronic messages. Byvirtue of the IRES element, each open reading frame may be accessible toribosomes for efficient translation. Multiple genes can be efficientlyexpressed using a single promoter/enhancer to transcribe a singlemessage. An alternative system for co-expression of two proteins in genetherapy delivery vectors is the FMDV 2A system. The FMDV 2A systememploys a retroviral plasmid vector in which two genes may be linked toa nucleotide sequence encoding the 2A sequence from the picornavirusfoot-and-mouth disease virus. Transcription and translation gives riseto a bicistronic mRNA and two independent protein products.

Any heterologous open reading frame can be linked to IRES elements. Thismay include genes for secreted proteins, multi-subunit proteins, encodedby independent genes, intracellular or membrane-bound proteins andselectable markers. In this way, expression of several proteins can besimultaneously engineered into a cell with a single construct and asingle selectable marker.

In some aspects, the recombinant virus and/or plasmid used to generaterecombinant virus, comprise a polynucleotide encoding a human mCoChopprotein or a functional fragment thereof.

In some aspects, the recombinant virus and/or plasmid used to generaterecombinant virus, comprise a regulatory nucleic acid fragment that iscapable of directing selective expression of the mCoChop protein in aneye cell.

In some aspects, the recombinant virus and/or plasmid used to generaterecombinant virus, may comprise one or more untranslated regions (UTR)or sequences. Generally, any suitable UTR sequence may be used for thedesired optimal expression of the transgene (i.e. mCoChop). For example,in some cases, UTR regions or sequences may comprise native sequences.In some cases, UTR sequences may be sequences as found upstream (5′ UTR)or downstream (3′UTR) of the human mCoChop gene as found in humangenomic sequence or portions thereof. In other cases, UTR sequences maycomprise non-native sequences, such as found upstream or downstream ofgenes other than mCoChop or comprise sequences further comprising acombination of one or more UTR sequence elements as further describedherein. In some cases, only a 5′ UTR sequence is used. In some cases,only a 3′ UTR sequence is used. In some cases, no UTR sequences areused.

A UTR sequence may comprise a length of 1-10 bp, 10-20 bp, 20-50 bp,50-100 bp, 100-500 bp, 500 bp-1 Kb, 1 Kb-2 Kb, 2 Kb-3 Kb, 3 Kb-4 Kb, 4Kb-5 Kb, 5 Kb-6 Kb, 6 Kb-7 Kb, 7 Kb-8 Kb, 8 Kb-9 Kb, and 9 Kb-10 Kb inlength. A UTR sequence may comprise a length of at least 1 bp, 2 bp, 3bp, 4 bp, 5 bp, 6 bp, 7 bp, 8 bp, 9 bp, 10 bp, 20 bp, 30 bp, 40 bp, 50bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500 bp,600 bp, 700 bp, 800 bp, 900 bp, 1 Kb, 2 Kb, 3 Kb, 4 Kb, 5 Kb, 6 Kb, 7Kb, 8 Kb, 9 Kb, and 10 Kb in length. A UTR sequence may comprise alength of at most 1 bp, 2 bp, 3 bp, 4 bp, 5 bp, 6 bp, 7 bp, 8 bp, 9 bp,10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp,200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1 Kb, 2Kb, 3 Kb, 4 Kb, 5 Kb, 6 Kb, 7 Kb, 8 Kb, 9 Kb, and 10 Kb in length.

In some cases, variations of either the 5′UTR and/or 3′UTR may beoptimized for a desired level of protein expression. In some cases,3′UTR sequences may be optimized for various parameters affectingprotein expression, including but not limited to mRNA half-life of thetransgene in the cell, stability or secondary structure of the mRNA ofthe transgene or conditional regulation (e.g. binding of various factorsto modulate translation). For example, the 3′UTR sequence maybe alteredto increase the half-life of the mRNA transcript of the transgene, whichmay result in increased protein expression. In some cases, the 3′UTRsequence maybe altered to decrease the half-life of the mRNA transcriptof the transgene, which may result in decreased protein expression.

Generally, 3′ UTRs sequences may comprise various sequence elements. Thepresent disclosure provides for 3′ UTR sequences that may include butare not limited to sequence elements such as one or more polyadenylationsignals, linker sequences, spacer sequences, SECIS elements, AU-rich orARE sequences or miRNA or RNAi binding sequences, transcriptionterminator sequences, 3′ termination sequences or variants and/orcombinations thereof.

In some cases, 5′UTR sequences may be optimized for various parametersaffecting protein expression, including but not limited to mRNAhalf-life of the transgene in the cell, stability or secondary structureof the mRNA of the transgene or transcriptional regulation. For example,the 5′UTR sequences may be altered to increase translation efficiency ofmRNA transcript of the transgene, which may result in increased proteinexpression. In some cases, the 5′UTR sequences maybe altered to decreasetranslation efficiency of mRNA transcript of the transgene, which mayresult in decreased protein expression.

Generally, 5′ UTRs sequences may comprise various sequence elements. Thepresent disclosure provides for 5′ UTR sequences that may include butare not limited to sequence elements such as one or more ribosomebinding sites (RBS), linker sequences, spacer sequences, regulatorysequences, regulatory response elements, riboswitches, sequences thatpromote or inhibit translation initiation, regulatory sequences for mRNAtransport or variants and/or combinations thereof.

In some aspects, the recombinant virus and/or plasmid used to generaterecombinant virus, may comprise one or more linker or spacer sequences.As described herein, linker sequence or spacer sequence may be usedinterchangeably. Generally, a linker sequence or spacer sequence may beany suitable sequence used to create a non-contiguous sequence betweenat least two sequence elements. Generally, any suitable linker or spacersequence may be used to create non-contiguous sequences. For example, insome cases, linker sequences may be randomly generated sequence. In somecases, linker sequence may be non-specific sequence optimized to preventformation of secondary structure or intramolecular interactions that mayadversely affect protein expression. In some cases, linker sequences maycomprise any additional functional sequence elements, including but notlimited to introns, regulatory sequences, enhancers or the like.Functional elements in linker sequences may be used for the desiredoptimal production of virus and/or expression of transgene expression.In some cases, linker sequences are cloning sites, remnants of priorcloning sites or other non-significant sequences and the insertion ofsuch linkers between any two sequence elements is optional.

A linker sequence may comprise a length of 1-10 bp, 10-20 bp, 20-50 bp,50-100 bp, 100-500 bp, 500 bp-1 Kb, 1 Kb-2 Kb, 2 Kb-3 Kb, 3 Kb-4 Kb, 4Kb-5 Kb, 5 Kb-6 Kb, 6 Kb-7 Kb, 7 Kb-8 Kb, 8 Kb-9 Kb, and 9 Kb-10 Kb inlength. A linker sequence may comprise a length of at least 1 bp, 2 bp,3 bp, 4 bp, 5 bp, 6 bp, 7 bp, 8 bp, 9 bp, 10 bp, 20 bp, 30 bp, 40 bp, 50bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500 bp,600 bp, 700 bp, 800 bp, 900 bp, 1 Kb, 2 Kb, 3 Kb, 4 Kb, 5 Kb, 6 Kb, 7Kb, 8 Kb, 9 Kb, and 10 Kb in length. A linker sequence may comprise alength of at most 1 bp, 2 bp, 3 bp, 4 bp, 5 bp, 6 bp, 7 bp, 8 bp, 9 bp,10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp,200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1 Kb, 2Kb, 3 Kb, 4 Kb, 5 Kb, 6 Kb, 7 Kb, 8 Kb, 9 Kb, and 10 Kb in length.

In some aspects, the recombinant virus comprises inverted terminalrepeat (ITR) sequences used for packaging the recombinant geneexpression cassette into the virion of the viral vector. In some cases,the ITR is from adeno-associated virus (AAV). In some cases, the ITR isfrom AAV serotype 2.

In some aspects, the recombinant virus and/or plasmid used to generaterecombinant virus comprises nucleic acid elements in the followingorder: a) a first ITR sequence; b) an enhancer sequence; c) a promotersequence; d) a first exon sequence; e) an intron sequence; f) a secondexon sequence; g) a sequence encoding mCoChop; h) a poly A sequence; andi) a second ITR sequence. In some aspects of the recombinant virusand/or plasmid used to generate the recombinant virus, the promotersequence comprises a promoter/enhancer sequence. In some aspects, thesequence encoding mCoChop comprises a sequence encoding human mCoChopprotein or a functional fragment thereof. In other aspects, the plasmidused to generate the recombinant virus further comprises an origin ofreplication sequence. In some aspects, the plasmid further comprises asequence for an antibiotic resistance gene.

Pharmaceutical Compositions

A pharmaceutical composition is a formulation containing one or moreactive ingredients as well as one or more excipients, carriers,stabilizers or bulking agents, which is suitable for administration to ahuman patient to achieve a desired diagnostic result or therapeutic orprophylactic effect. For storage stability and convenience of handling,a pharmaceutical composition can be formulated as a lyophilized (i.e.freeze dried) or vacuum dried powder which can be reconstituted withsaline or water prior to administration to a patient. Alternately, thepharmaceutical composition can be formulated as an aqueous solution. Apharmaceutical composition can contain a proteinaceous activeingredient. Various excipients, such as albumin and gelatin have beenused with differing degrees of success to try and stabilize a proteinactive ingredient present in a pharmaceutical composition. Additionally,cryoprotectants such as alcohols have been used to reduce proteindenaturation under the freezing conditions of lyophilization.

Pharmaceutical compositions suitable for internal use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersion. For intravenous administration, suitable carriers includephysiological saline, bacteriostatic water, or phosphate buffered saline(PBS). In all cases, the composition must be sterile and should be fluidto the extent that easy syringability exists. It must be stable underthe conditions of manufacture and storage and must be preserved againstthe contaminating action of microorganisms such as bacteria and fungi.The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants such as polysorbates (Tween™), sodium dodecyl sulfate(sodium lauryl sulfate), lauryl dimethyl amine oxide,cetyltrimethylammonium bromide (CTAB), polyethoxylated alcohols,polyoxyethylene sorbitan, octoxynol (Triton X100™),N,N-dimethyldodecylamine-N-oxide, hex adecyltrimethylammonium bromide(HTAB), polyoxyl 10 lauryl ether, Brij 721™, bile salts (sodiumdeoxycholate, sodium cholate), pluronic acids (F-68, F-127), polyoxylcastor oil (Cremophor™) nonylphenol ethoxylate (Tergitol™),cyclodextrins and, ethylbenzethonium chloride (Hyamine™) Prevention ofthe action of microorganisms can be achieved by various antibacterialand antifungal agents, for example, parabens, chlorobutanol, phenol,ascorbic acid, thimerosal, and the like. In many cases, it will bepreferable to include isotonic agents, for example, sugars, polyalcoholssuch as manitol, sorbitol, sodium chloride in the composition. Prolongedabsorption of the internal compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, aluminum monostearate and gelatin.

Sterile solutions can be prepared by incorporating the active compoundin the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

In one aspect, active compounds are prepared with carriers that willprotect the compound against rapid elimination from the body, such as acontrolled release formulation, including implants and microencapsulateddelivery systems. Biodegradable, biocompatible polymers can be used,such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid,collagen, polyorthoesters, and polylactic acid. Methods for preparationof such formulations will be apparent to those skilled in the art. Thematerials can also be obtained commercially. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811, incorporated by reference herein.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Pharmaceutical compositions of the present disclosure comprise, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that comprise, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

Certain compositions of the present disclosure also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The co-administration of a nucleic acid and a carriercompound, generally with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extra circulatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate oligonucleotide in hepatic tissue can be reduced whenit is co-administered with polyinosinic acid, dextran sulphate,polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′disulfonicacid (Miyao et al., Antisense Res. Dev., 1995, 5, 115-121; Takakura etal., Antisense & Nucl. Acid Drug Dev., 1996, 6, 177-183).

The vector or recombinant viruses (virions) can be incorporated intopharmaceutical compositions for administration to mammalian patients,particularly humans. The vector or virions can be formulated innontoxic, inert, pharmaceutically acceptable aqueous carriers,preferably at a pH ranging from 3 to 8, more preferably ranging from 6to 8, most preferably ranging from 6.8 to 7.2. Such sterile compositionswill comprise the vector or virion containing the nucleic acid encodingthe therapeutic molecule dissolved in an aqueous buffer having anacceptable pH upon reconstitution.

In some aspects, the pharmaceutical compositions provided hereincomprise a therapeutically effective amount of a vector or virion inadmixture with a pharmaceutically acceptable carrier and/or excipient,for example saline, phosphate buffered saline, phosphate and aminoacids, polymers, polyols, sugar, buffers, preservatives and otherproteins. Exemplary amino acids, polymers and sugars and the like areoctylphenoxy polyethoxy ethanol compounds, polyethylene glycolmonostearate compounds, polyoxyethylene sorbitan fatty acid esters,sucrose, fructose, dextrose, maltose, glucose, mannitol, dextran,sorbitol, inositol, galactitol, xylitol, lactose, trehalose, bovine orhuman serum albumin, citrate, acetate, Ringer's and Hank's solutions,cysteine, arginine, carnitine, alanine, glycine, lysine, valine,leucine, polyvinylpyrrolidone, polyethylene and glycol. Preferably, thisformulation is stable for at least 14 months at −60° C.

In some aspects, the pharmaceutical composition provided hereincomprises a buffer, such as phosphate buffered saline (PBS) or sodiumphosphate/sodium sulfate, tris buffer, glycine buffer, sterile water andother buffers known to the ordinarily skilled artisan such as thosedescribed by Good et al. (1966) Biochemistry 5:467. Preferredpharmaceutical composition contains sodium phosphate, sodium chlorideand sorbital. Most preferred pharmaceutical composition contains 10 mMsodium phosphate, 350 mM sodium chloride and 5% (v/v) sorbital. The pHof the buffer in which the pharmaceutical composition comprising themCoChop contained in the adenoviral vector delivery system, may be inthe range of 6.5 to 7.75, 6.5 to 7.5, 6.8 to 7.4 or 6.8 to 7.2.

In some aspects, the pharmaceutical composition provided hereincomprises substances which increase the viscosity of the suspension,such as sodium carboxymethyl cellulose, sorbitol, or dextran, in theamount about 1-10 percent, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10percent (v/v). Preferably the sorbitol is about 3-6% (v/v), mostpreferably the sorbitol is about 5%. (v/v).

Prior to administration the pharmaceutical composition is free ofcomponents used during the production, e.g., culture components, hostcell protein, host cell DNA, plasmid DNA and substantially free ofmycoplasm, endotoxin, and microbial contamination. Preferably, thepharmaceutical composition has less than 10, 5, 3, 2, or 1 CFU/swab.Most preferably composition has 0 CFU/swab. The endotoxin level in thepharmaceutical composition is less than 20 EU/mL, less than 10 EU/mL orless than 5 EU/mL.

The pharmaceutical composition must have sufficiently full capsid priorto administration. The pharmaceutical composition has at least 50%, atleast 60%, at least 70%, at least 80% or greater full capsids.

Kits

Compositions and reagents useful for the present disclosure may bepackaged in kits to facilitate application of the present disclosure. Insome aspects, the present method provides for a kit comprising arecombinant nucleic acid of the disclosure. In some aspects, the presentmethod provides for a kit comprising a recombinant virus of thedisclosure. The instructions could be in any desired form, including butnot limited to, printed on a kit insert, printed on one or morecontainers, as well as electronically stored instructions provided on anelectronic storage medium, such as a computer readable storage medium.Also optionally included is a software package on a computer readablestorage medium that permits the user to integrate the information andcalculate a control dose.

In another aspect, the present disclosure provides a kit comprising thepharmaceutical compositions provided herein. In yet another aspect, thedisclosure provides kits in the treatment of diseases.

In one aspect, a kit comprises: (a) a recombinant virus provided herein,and (b) instructions to administer to cells or an individual atherapeutically effective amount of the recombinant virus. In someaspects, the kit may comprise pharmaceutically acceptable salts orsolutions for administering the recombinant virus. Optionally, the kitcan further comprise instructions for suitable operational parameters inthe form of a label or a separate insert. For example, the kit may havestandard instructions informing a physician or laboratory technician toprepare a dose of recombinant virus.

Optionally, the kit may further comprise a standard or controlinformation so that a patient sample can be compared with the controlinformation standard to determine if the test amount of recombinantvirus is a therapeutic amount Optionally, the kit could further comprisedevices for administration, such as a syringe, filter needle, extensiontubing, cannula, and subretinal injector.

Recombinant viruses may be generated by any suitable means. The methodsand compositions and of the disclosure provide for generation ofrecombinant virus through various means, including the use of transgeniccells, which may include mammalian cells, insect cells, animal cells orfungal cells.

For example, in some aspects, recombinant viruses may be generatedthrough transfection of insect cells via recombinant baculovirus. Insome cases, recombinant baculovirus may be generated as an intermediate,whereby the baculovirus may contain sequences necessary for thegeneration of other viruses such as AAV or rAAV2 viruses. In some casesone or more baculoviruses may be used in the generation of recombinantviruses used for the composition and methods of treatment of thisdisclosure. In some cases insect cells such as Sf9, High-Five or Sf21cell lines may be used. In some cases, cell lines may be generated usingtransient methods (i.e. infection with not stably integratedtransgenes). In other cases, cell lines may be generated through thegeneration of stable cell lines (i.e. infection with transgenes stablyintegrated into the host cell genome.) In other aspects, thepharmaceutical composition provided herein is manufactured usingadherent human embryonic kidney 293 (HEK293) cells. In an alternativeaspect, the pharmaceutical composition provided herein is manufacturedusing suspension-adapted HEK293 cells. In another aspect, thepharmaceutical composition provided herein is manufactured using thebaculovirus expression system (BYES) in insect cells. In some aspects,the vector is produced using herpes-helper virus. In some aspects, thevector is produced using producer-clone methods. In some aspects, thevector is produced using Ad-AAV.

Generally, any suitable method may be used in the biochemicalpurification of recombinant viruses for use in a pharmaceuticalcomposition as described herein. Recombinant viruses may be harvesteddirectly from cells, or from the culture media surrounding host cells.Virus may be purified using various biochemical means, such as gelfiltration, filtration, chromatography, affinity purification, gradientultracentrifugation, or size exclusion methods. Recombinant virus may betested for content (i.e., identity), purity, or potency (i.e., activity)using any suitable means, before formulation into a pharmaceuticalcomposition. Method may include but are not limited to immunoassays,ELISA, SDS-PAGE, western blot, Northern blot, Southern blot or PCR,HUVEC assays and the like.

Methods of Treatment

The ocular disorders for which the present mCoChop proteins and nucleicacids, and the resulting ChR proteins, are intended and may be used toimprove one or more parameters of vision include, but are not limitedto, developmental abnormalities that affect both anterior and posteriorsegments of the eye. Anterior segment disorders include glaucoma,cataracts, corneal dystrophy, keratoconus. Posterior segment disordersinclude blinding disorders caused by photoreceptor malfunction and/ordeath caused by retinal dystrophies and degenerations. Retinal disordersinclude congenital stationary night blindness, macular degeneration suchas age-related macular degeneration, congenital cone dystrophies, and alarge group of retinitis-pigmentosa (RP)-related disorders. Thesedisorders include genetically pre-disposed death of photoreceptor cells,rods and cones in the retina, occurring at various ages. Among those aresevere retinopathies, such as subtypes of RP itself that progresses withage and causes blindness in childhood and early adulthood andRP-associated diseases, such as genetic subtypes of LCA, whichfrequently results in loss of vision during childhood, as early as thefirst year of life. The latter disorders are generally characterized bysevere reduction, and often complete loss of photoreceptor cells, rodsand cones. (Trabulsi, E I, ed., Genetic Diseases of the Eye, OxfordUniversity Press, N Y, 1998).

In particular, the mCoChop and ChR proteins of the present inventionuseful for the treatment and/or restoration of at least partial visionto subjects that have lost vision due to ocular disorders, such asRPE-associated retinopathies, which are characterized by a long-termpreservation of ocular tissue structure despite loss of function and bythe association between function loss and the defect or absence of anormal gene in the ocular cells of the subject. A variety of such oculardisorders are known, such as childhood onset blinding diseases,retinitis pigmentosa, macular degeneration, and diabetic retinopathy, aswell as ocular blinding diseases known in the art. It is anticipatedthat these other disorders, as well as blinding disorders of presentlyunknown causation which later are characterized by the same descriptionas above, may also be successfully treated by the CoChop and ChRproteins of the present invention. Thus, the particular ocular disordertreated by the present invention may include the above-mentioneddisorders and a number of diseases which have yet to be socharacterized.

In particular embodiments, the present disclosure provides a method fortreating retinal degenerative diseases, comprising administering apharmaceutically effective amount of the pharmaceutical compositionsprovided herein to a subject in need of such treatment. Preferably, theretinal degenerative disease is retinitis pigmentosa or age-relatedmacular degeneration (AMD), wet-AMD, dry-AMD. Additionally, otherdiseases and disorders that are the direct result of retinaldegenerative diseases are also treated by the method of the invention.

In some embodiments, dry AMD may be treated. In some cases, dry AMD maybe referred to as central geographic atrophy, characterized by atrophyof the retinal pigment epithelial later below the retina and subsequentloss of photoreceptors in the central part of the eye. The compositionand methods of this disclosure provide for the treatment of any and allforms of AMD.

In another aspect, the present disclosure provides a method forprophylactic treatment of AMD or retinitis pigmentosa as describedherein, comprising administering a pharmaceutically effective amount ofthe pharmaceutical compositions provided herein to a human subject inneed of such treatment. The present disclosure may be used to treatpatients at risk of developing AMD, or presenting early symptoms of thedisease. The present disclosure may be used to treat patients at risk ofdeveloping MD, or presenting early symptoms of the disease, such asthose individuals having a retinal degenerative disease. This mayinclude treatment of eyes either simultaneously or sequentially.Simultaneous treatment may mean that the treatment is administered toeach eye at the same time or that both eyes are treated during the samevisit to a treating physician or other healthcare provider. It has beendocumented that patients have a higher risk of developing AMD in ahealthy fellow eye of an eye that presents symptoms of AMD, or inpatients who have a genetic predisposition toward developing AMD. Thepresent disclosure can be used as a prophylactic treatment in preventionof AMD in the fellow eye.

In some embodiments, mutant CoChop compositions (e.g. nucleotides,polypeptides, cells expressing said polypeptides or containing saidnucleotides, pharmaceutical compositions, etc.) disclosed herein areadministered to a patient. In some embodiments, mutant CoChopcompositions created using the methods ameliorate, or delay the onset ofa disease or disorder. In some embodiments, the disease or disorder is adegenerative disease or disorder. In some embodiments, the disease ordisorder is an ocular disorder. In some embodiments, the ocular disorderis AMD, macular degeneration or retinitis pigmentosa. In someembodiments, the disease or disorder is injury, brain damage, spinalcord injury, epilepsy, a metabolic disorder, a cardiac dysfunction,vison loss, blindness, deafness, hearing loss or neurological condition.In some embodiments, mutant CoChop compositions disclosed herein areadministered to a patient to restore vision loss. In some embodiments,mutant CoChop compositions disclosed herein are administered to apatient to prevent, delay, or ameliorate vision loss.

In some embodiments, the mutant CoChop compositions disclosed herein areadministered once to a patient. In some embodiments, the vectors,nucleic acids, or cells disclosed herein are administered about 2 times,about 3 time, about 4 times, about 5 times, about 6 times, about 7times, about 8 times, about 9 times, about 10 times, about 20 times,about 40 times, or more to a patient. Mutant CoChop compositionsdisclosed herein are administered until disease or disorder symptomsimprove.

In some embodiments, administration of the mutant CoChop compositionsdisclosed herein improves, prevents, delays, or ameliorates vision lossin a treated patient compared to an untreated patient or the samepatient before treatment. In some embodiments, administration of themutant CoChop compositions disclosed herein improves, prevents, delays,or ameliorates vision loss in a treated patient between day 1 and year10. In some embodiments, administration of administration of the mutantCoChop compositions disclosed herein improves, prevents, delays, orameliorates vision loss at about day 1, about day 2, about day 3, aboutday 4, about day 5, about day 6, about week 1, about week 2, about week3, about week 4, about week 5, about week 6, about week 7, about week 8,about week 9, about week 10, about week 20, about week 30, about week40, about week 50, about week 60, about week 70, about week 80, aboutweek 90, about week 100, about year 1, about year 2, or about year 3compared with vision loss in an untreated patient or the same patientbefore treatment. In some embodiments, administration of the mutantCoChop compositions disclosed herein improves, prevents, delays, orameliorates vision loss for about 1 day, about 1 week, about 1 month,about 2 months, about 3 months, about 4 months, about 5 months, about 6months, about 1 year, about 2 years, about 5 years, or about 10 years,or more compared with vision loss in an untreated patient or the samepatient before treatment.

In some embodiments, vision loss is decreased by about 1%, about 5%,about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about70%, about 80%, about 90%, or about 100% compared with controls orpatients treated with other compositions. In some embodiments,administration of the mutant CoChop compositions improves, prevents,ameliorates, or delays vision loss by about 1%, about 5%, about 10%,about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about80%, about 90%, or about 100% at about day 1, about day 2, about day 3,about day 4, about day 5, about day 6, about week 1, about week 2, aboutweek 3, about week 4, about week 5, about week 6, about week 7, aboutweek 8, about week 9, about week 10, about week 20, about week 30, aboutweek 40, about week 50, about week 60, about week 70, about week 80,about week 90, about week 100, about year 1, about year 2, or about year3 compared with controls or patients treated with other compositions. Insome embodiments, administration of the mutant CoChop compositionsdisclosed herein improves, prevents, ameliorates, or delays vision lossby about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about50%, about 60%, about 70%, about 80%, about 90%, or about 100% for about1, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days,about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1month, about 2 months, about 3 months, about 4 months, about 5 months,about 6 months, about 1 year, about 2 years, about 5 years, or about 10years or more compared with controls or patients treated with othermethods.

In some embodiments, administration of the mutant CoChop compositionsdisclosed herein increases light sensitivity in a treated patientcompared to an untreated patient or the same patient before treatment.In some embodiments, administration of the mutant CoChop compositionsdisclosed herein increases light sensitivity in a treated patientbetween day 1 and year 10. In some embodiments, administration ofadministration of the mutant CoChop compositions disclosed hereinincreases light sensitivity at about day 1, about day 2, about day 3,about day 4, about day 5, about day 6, about week 1, about week 2, aboutweek 3, about week 4, about week 5, about week 6, about week 7, aboutweek 8, about week 9, about week 10, about week 20, about week 30, aboutweek 40, about week 50, about week 60, about week 70, about week 80,about week 90, about week 100, about year 1, about year 2, or about year3 compared with light sensitivity an untreated patient or the samepatient before treatment. In some embodiments, administration of themutant CoChop compositions disclosed herein increases light sensitivityfor about 1 day, about 1 week, about 1 month, about 2 months, about 3months, about 4 months, about 5 months, about 6 months, about 1 year,about 2 years, about 5 years, or about 10 years, or more compared withlight sensitivity in an untreated patient or the same patient beforetreatment.

In some embodiments, light sensitivity is increased by about 1%, about5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%,about 70%, about 80%, about 90%, or about 100% compared with controls orpatients treated with other compositions. In some embodiments,administration of the mutant CoChop compositions increases lightsensitivity by about 1%, about 5%, about 10%, about 20%, about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, orabout 100% at about day 1, about day 2, about day 3, about day 4, aboutday 5, about day 6, about week 1, about week 2, about week 3, about week4, about week 5, about week 6, about week 7, about week 8, about week 9,about week 10, about week 20, about week 30, about week 40, about week50, about week 60, about week 70, about week 80, about week 90, aboutweek 100, about year 1, about year 2, or about year 3 compared withcontrols or patients treated with other compositions. In someembodiments, administration of the mutant CoChop compositions disclosedherein increases light sensitivity by about 1%, about 5%, about 10%,about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about80%, about 90%, or about 100% for about 1, about 2 days, about 3 days,about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks,about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3months, about 4 months, about 5 months, about 6 months, about 1 year,about 2 years, about 5 years, or about 10 years or more compared withcontrols or patients treated with other methods.

In some embodiments, administration of the mutant CoChop compositionsdisclosed herein decreases the light intensity required to elicit aphotocurrent in a treated patient compared to an untreated patient orthe same patient before treatment. In some embodiments, administrationof the mutant CoChop compositions disclosed herein decreases the lightintensity required to elicit a photocurrent in a treated patient betweenday 1 and year 10. In some embodiments, administration of administrationof the mutant CoChop compositions disclosed herein decreases the lightintensity required to elicit a photocurrent at about day 1, about day 2,about day 3, about day 4, about day 5, about day 6, about week 1, aboutweek 2, about week 3, about week 4, about week 5, about week 6, aboutweek 7, about week 8, about week 9, about week 10, about week 20, aboutweek 30, about week 40, about week 50, about week 60, about week 70,about week 80, about week 90, about week 100, about year 1, about year2, or about year 3 compared with the light intensity required to elicita photocurrent in an untreated patient or the same patient beforetreatment. In some embodiments, administration of the mutant CoChopcompositions disclosed herein decreases the light intensity required toelicit a photocurrent for about 1 day, about 1 week, about 1 month,about 2 months, about 3 months, about 4 months, about 5 months, about 6months, about 1 year, about 2 years, about 5 years, or about 10 years,or more compared with the light intensity required to elicit aphotocurrent in an untreated patient or the same patient beforetreatment.

In some embodiments, the light intensity required to elicit aphotocurrent is decreased by about 1%, about 5%, about 10%, about 20%,about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about90%, or about 100% compared with controls or patients treated with othercompositions. In some embodiments, administration of the mutant CoChopcompositions decreases the light intensity required to elicit aphotocurrent by about 1%, about 5%, about 10%, about 20%, about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, orabout 100% at about day 1, about day 2, about day 3, about day 4, aboutday 5, about day 6, about week 1, about week 2, about week 3, about week4, about week 5, about week 6, about week 7, about week 8, about week 9,about week 10, about week 20, about week 30, about week 40, about week50, about week 60, about week 70, about week 80, about week 90, aboutweek 100, about year 1, about year 2, or about year 3 compared withcontrols or patients treated with other compositions. In someembodiments, administration of the mutant CoChop compositions disclosedherein decreases the light intensity required to elicit a photocurrentby about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about50%, about 60%, about 70%, about 80%, about 90%, or about 100% for about1, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days,about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1month, about 2 months, about 3 months, about 4 months, about 5 months,about 6 months, about 1 year, about 2 years, about 5 years, or about 10years or more compared with controls or patients treated with othermethods.

In some embodiments, administration of the mutant CoChop compositionsdisclosed herein increases ion flux and/or proton flux in a treatedpatient compared to an untreated patient or the same patient beforetreatment. In some embodiments, administration of the mutant CoChopcompositions disclosed herein increases ion flux and/or proton flux in atreated patient between day 1 and year 10. In some embodiments,administration of administration of the mutant CoChop compositionsdisclosed herein increases ion flux and/or proton flux at about day 1,about day 2, about day 3, about day 4, about day 5, about day 6, aboutweek 1, about week 2, about week 3, about week 4, about week 5, aboutweek 6, about week 7, about week 8, about week 9, about week 10, aboutweek 20, about week 30, about week 40, about week 50, about week 60,about week 70, about week 80, about week 90, about week 100, about year1, about year 2, or about year 3 compared with ion flux and/or protonflux an untreated patient or the same patient before treatment. In someembodiments, administration of the mutant CoChop compositions disclosedherein increases ion flux and/or proton flux for about 1 day, about 1week, about 1 month, about 2 months, about 3 months, about 4 months,about 5 months, about 6 months, about 1 year, about 2 years, about 5years, or about 10 years, or more compared with ion flux and/or protonflux in an untreated patient or the same patient before treatment.

In some embodiments, ion flux and/or proton flux is increased by about1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%,about 60%, about 70%, about 80%, about 90%, or about 100% compared withcontrols or patients treated with other compositions. In someembodiments, administration of the mutant CoChop compositions increasesion flux and/or proton flux by about 1%, about 5%, about 10%, about 20%,about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about90%, or about 100% at about day 1, about day 2, about day 3, about day4, about day 5, about day 6, about week 1, about week 2, about week 3,about week 4, about week 5, about week 6, about week 7, about week 8,about week 9, about week 10, about week 20, about week 30, about week40, about week 50, about week 60, about week 70, about week 80, aboutweek 90, about week 100, about year 1, about year 2, or about year 3compared with controls or patients treated with other compositions. Insome embodiments, administration of the mutant CoChop compositionsdisclosed herein increases ion flux and/or proton flux by about 1%,about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about60%, about 70%, about 80%, about 90%, or about 100% for about 1, about 2days, about 3 days, about 4 days, about 5 days, about 6 days, about 1week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about2 months, about 3 months, about 4 months, about 5 months, about 6months, about 1 year, about 2 years, about 5 years, or about 10 years ormore compared with controls or patients treated with other methods.

In some embodiments, administration of the mutant CoChop compositionsdisclosed herein increases visual evoked potential in the visual cortexin a treated patient compared to an untreated patient or the samepatient before treatment. In some embodiments, administration of themutant CoChop compositions disclosed herein increases visual evokedpotential in the visual cortex in a treated patient between day 1 andyear 10. In some embodiments, administration of administration of themutant CoChop compositions disclosed herein increases visual evokedpotential in the visual cortex at about day 1, about day 2, about day 3,about day 4, about day 5, about day 6, about week 1, about week 2, aboutweek 3, about week 4, about week 5, about week 6, about week 7, aboutweek 8, about week 9, about week 10, about week 20, about week 30, aboutweek 40, about week 50, about week 60, about week 70, about week 80,about week 90, about week 100, about year 1, about year 2, or about year3 compared with the visual evoked potential in the visual cortex in anuntreated patient or the same patient before treatment. In someembodiments, administration of the mutant CoChop compositions disclosedherein increases visual evoked potential in the visual cortex for about1 day, about 1 week, about 1 month, about 2 months, about 3 months,about 4 months, about 5 months, about 6 months, about 1 year, about 2years, about 5 years, or about 10 years, or more compared with thevisual evoked potential in the visual cortex in an untreated patient orthe same patient before treatment.

In some embodiments, the visual evoked potential in the visual cortex isincreased by about 1%, about 5%, about 10%, about 20%, about 30%, about40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about100% compared with controls or patients treated with other compositions.In some embodiments, administration of the mutant CoChop compositionsincreases visual evoked potential in the visual cortex by about 1%,about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about60%, about 70%, about 80%, about 90%, or about 100% at about day 1,about day 2, about day 3, about day 4, about day 5, about day 6, aboutweek 1, about week 2, about week 3, about week 4, about week 5, aboutweek 6, about week 7, about week 8, about week 9, about week 10, aboutweek 20, about week 30, about week 40, about week 50, about week 60,about week 70, about week 80, about week 90, about week 100, about year1, about year 2, or about year 3 compared with controls or patientstreated with other compositions. In some embodiments, administration ofthe mutant CoChop compositions disclosed herein increases visual evokedpotential in the visual cortex by about 1%, about 5%, about 10%, about20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%,about 90%, or about 100% for about 1, about 2 days, about 3 days, about4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3weeks, about 4 weeks, about 1 month, about 2 months, about 3 months,about 4 months, about 5 months, about 6 months, about 1 year, about 2years, about 5 years, or about 10 years or more compared with controlsor patients treated with other methods.

In some embodiments, administration of the mutant CoChop compositionsdisclosed herein reduce disease or disorder symptoms in a treatedpatient compared to an untreated patient or the same patient beforetreatment. In some embodiments, the disease or disorder symptoms aremeasured in a treated patient between day 1 and year 10. In someembodiments, administration of the mutant CoChop compositions disclosedherein reduces a disease or disorder symptom at about day 1, about day2, about day 3, about day 4, about day 5, about day 6, about week 1,about week 2, about week 3, about week 4, about week 5, about week 6,about week 7, about week 8, about week 9, about week 10, about week 20,about week 30, about week 40, about week 50, about week 60, about week70, about week 80, about week 90, about week 100, about year 1, aboutyear 2, or about year 3 compared with the disease or disorder symptom inan untreated patient or the same patient before treatment. In someembodiments, administration of the mutant CoChop compositions disclosedherein reduces a disease or disorder symptom for about 1 day, about 2days, about 3 days, about 4 days, about 5 days, about 6 days, about 1week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about2 months, about 3 months, about 4 months, about 5 months, about 6months, about 1 year, about 2 years, about 5 years, or about 10 years,or more compared with the disease or disorder symptom in an untreatedpatient or the same patient before treatment.

In some embodiments, the disease or disorder symptom is reduced by about1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%,about 60%, about 70%, about 80%, about 90%, or about 100% compared withthe disease or disorder symptom in an untreated patient or the samepatient before treatment. In some embodiments, administration of themutant CoChop compositions disclosed herein reduces the disease ordisorder symptom by about 1%, about 5%, about 10%, about 20%, about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, orabout 100% at about day 1, about day 2, about day 3, about day 4, aboutday 5, about day 6, about week 1, about week 2, about week 3, about week4, about week 5, about week 6, about week 7, about week 8, about week 9,about week 10, about week 20, about week 30, about week 40, about week50, about week 60, about week 70, about week 80, about week 90, aboutweek 100, about year 1, about year 2, or about year 3 compared with thedisease or disorder symptom in an untreated patient or the same patientbefore treatment. In some embodiments, administration of the mutantCoChop compositions disclosed herein reduces the disease or disordersymptom by about 1%, about 5%, about 10%, about 20%, about 30%, about40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about100% for about 1 day, about 2 days, about 3 days, about 4 days, about 5days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4weeks, about 1 month, about 2 months, about 3 months, about 4 months,about 5 months, about 6 months, about 1 year, about 2 years, about 5years, or about 10 years or more compared with the disease or disordersymptom in an untreated patient or the same patient before treatment.

In some embodiments, administration of the mutant CoChop compositionsdisclosed herein reduce symptoms of AMD in a treated patient compared toan untreated patient or the same patient before treatment. In someembodiments, the symptom is blurred vision, decrease in visual acuity,partial loss of vision, and/or an inability to see in dim light. In someembodiments, the AMD symptoms are measured in a treated patient betweenday 1 and year 10. In some embodiments, administration of the mutantCoChop compositions disclosed herein reduces an AMD symptom at about day1, about day 2, about day 3, about day 4, about day 5, about day 6,about week 1, about week 2, about week 3, about week 4, about week 5,about week 6, about week 7, about week 8, about week 9, about week 10,about week 20, about week 30, about week 40, about week 50, about week60, about week 70, about week 80, about week 90, about week 100, aboutyear 1, about year 2, or about year 3 compared with the AMD symptom inan untreated patient or the same patient before treatment. In someembodiments, administration of the mutant CoChop compositions disclosedherein reduces an AMD symptom for about 1 day, about 2 days, about 3days, about 4 days, about 5 days, about 6 days, about 1 week, about 2weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months,about 3 months, about 4 months, about 5 months, about 6 months, about 1year, about 2 years, about 5 years, or about 10 years, or more comparedwith the AMD symptom in an untreated patient or the same patient beforetreatment.

In some embodiments, the AMD symptom is reduced by about 1%, about 5%,about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about70%, about 80%, about 90%, or about 100% compared with the AMD symptomin an untreated patient or the same patient before treatment. In someembodiments, administration of the mutant CoChop compositions disclosedherein reduces the AMD symptom by about 1%, about 5%, about 10%, about20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%,about 90%, or about 100% at about day 1, about day 2, about day 3, aboutday 4, about day 5, about day 6, about week 1, about week 2, about week3, about week 4, about week 5, about week 6, about week 7, about week 8,about week 9, about week 10, about week 20, about week 30, about week40, about week 50, about week 60, about week 70, about week 80, aboutweek 90, about week 100, about year 1, about year 2, or about year 3compared with the AMD symptom in an untreated patient or the samepatient before treatment. In some embodiments, administration of themutant CoChop compositions disclosed herein reduces the AMD symptom byabout 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about50%, about 60%, about 70%, about 80%, about 90%, or about 100% for about1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1month, about 2 months, about 3 months, about 4 months, about 5 months,about 6 months, about 1 year, about 2 years, about 5 years, or about 10years or more compared with the AMD symptom in an untreated patient orthe same patient before treatment.

The term “subject,” or “individual” or “patient” as used herein inreference to individuals having a disease or disorder or are suspectedof having a disease or disorder, and the like. Subject, individual orpatent may be used interchangeably in the disclosure and encompassmammals and non-mammals. Examples of mammals include, but are notlimited to, any member of the Mammalian class: humans, non-humanprimates such as chimpanzees, and other apes and monkey species; farmanimals such as cattle, horses, sheep, goats, swine; domestic animalssuch as rabbits, dogs, and cats; laboratory animals including rodents,such as rats, mice and guinea pigs, and the like. Examples ofnon-mammals include, but are not limited to, birds, fish and the like.In some aspects of the methods and compositions provided herein, themammal is a human.

Efficacy of the treatment will be established for example by evaluatingthe best corrected visual acuity of each eye. Visual acuity test areperformed using the Electronic visual acuity (EVA) ETDRS (EarlyTreatment for Diabetic Retinopathy Study) methodology, or low visionassessment of hand motion and light perception.

The term “vision” as used herein is defined as the ability of anorganism to usefully detect light as a stimulus for differentiation oraction. Vision is intended to encompass the following:

-   -   1. Light detection or perception—the ability to discern whether        or not light is present;    -   2. Light projection—the ability to discern the direction from        which a light stimulus is coming;    -   3. Resolution—the ability to detect differing brightness levels        (i.e., contrast) in a grating or letter target; and    -   4. Recognition—the ability to recognize the shape of a visual        target by reference to the differing contrast levels within the        target.        Thus, “vision” includes the ability to simply detect the        presence of light. The polypeptides and polynucleotides encoding        mutant CoChop of the present invention can be used to improve or        restore vision, wherein the improvement or restoration in vision        includes, for example, increases in light detection or        perception, increase in light sensitivity or photosensitivity in        response to a light stimulus, increase in the ability to discern        the direction from which a light stimulus is coming, increase in        the ability to detect differing brightness levels, increase in        the ability to recognize the shape of a visual target, and        increases in visual evoked potential or transmission from the        retina to the cortex. As such, improvement or restoration of        vision may or may not include full restoration of sight, i.e.,        wherein the vision of the patient treated with the present        invention is restored to the degree to the vision of a        non-affected individual. The visual recovery described in the        animal studies described below may, in human terms, place the        person on the low end of vision function by increasing one        aspect of vision (i.e., light sensitivity, or visual evoked        potential) without restoring full sight. Nevertheless, placement        at such a level would be a significant benefit because these        individuals could be trained in mobility and potentially in low        order resolution tasks which would provide them with a greatly        improved level of visual independence compared to total        blindness. Even basic light perception can be used by visually        impaired individuals, whose vision is improved using the present        compositions and methods, to accomplish specific daily tasks and        improve general mobility, capability, and quality of life.

The degree of restoration of vision can be determined through themeasurement of vision before, and preferably after, administering avector comprising, for example, DNA encoding CoChop. Vision can bemeasured using any of a number of methods well-known in the art ormethods not yet established. Vision, as improved or restored by thepresent invention, can be measured by any of the following visualresponses:

-   -   1. a light detection response by the subject after exposure to a        light stimulus—in which evidence is sought for a reliable        response of an indication or movement in the general direction        of the light by the subject individual when the light it is        turned on;    -   2. a light projection response by the subject after exposure to        a light stimulus in which evidence is sought for a reliable        response of indication or movement in the specific direction of        the light by the individual when the light is turned on;    -   3. light resolution by the subject of a light vs. dark patterned        visual stimulus, which measures the subject's capability of        resolving light vs dark patterned visual stimuli as evidenced        by:        -   a. the presence of demonstrable reliable optokinetically            produced nystagmoid eye movements and/or related head or            body movements that demonstrate tracking of the target (see            above) and/or        -   b. the presence of a reliable ability to discriminate a            pattern visual stimulus and to indicate such discrimination            by verbal or non-verbal means, including, for example            pointing, or pressing a bar or a button; or    -   4. electrical recording of a visual cortex response to a light        flash stimulus or a pattern visual stimulus, which is an        endpoint of electrical transmission from a restored retina to        the visual cortex, also referred to as the visual evoked        potential (VEP). Measurement may be by electrical recording on        the scalp surface at the region of the visual cortex, on the        cortical surface, and/or recording within cells of the visual        cortex.

Thus, improvement or restoration of vision, according to the presentinvention, can include, but is not limited to: increases in amplitude orkinetics of photocurents or electrical response in response to lightstimulus in the retinal cells, increases in light sensitivity (i.e.,lowering the threshold light intensity required for initiating aphotocurrent or electrical response in response to light stimulus,thereby requiring less or lower light to evoke a photocurrent) of theretinal cells, increases in number or amplitude of light-evoked spikingor spike firings, increases in light responses to the visual cortex,which includes increasing in visual evoked potential transmitted fromthe retina or retinal cells to the visual cortex or the brain.

Both in vitro and in vivo studies to assess the various parameters ofthe present invention may be used, including recognized animal models ofblinding human ocular disorders. Large animal models of humanretinopathy, e.g., childhood blindness, are useful. The examplesprovided herein allow one of skill in the art to readily anticipate thatthis method may be similarly used in treating a range of retinaldiseases.

While earlier studies by others have demonstrated that retinaldegeneration can be retarded by gene therapy techniques, the presentinvention demonstrates a definite physiological recovery of function,which is expected to generate or improve various parameters of vision,including behavioral parameters.

Behavioral measures can be obtained using known animal models and tests,for example performance in a water maze, wherein a subject in whomvision has been preserved or restored to varying extents will swimtoward light (Hayes, J M et al., 1993, Behav Genet 23:395-403).

In models in which blindness is induced during adult life or congenitalblindness develops slowly enough that the individual experiences visionbefore losing it, training of the subject in various tests may be done.In this way, when these tests are re-administered after visual loss totest the efficacy of the present compositions and methods for theirvision-restorative effects, animals do not have to learn the tasks denovo while in a blind state. Other behavioral tests do not requirelearning and rely on the instinctiveness of certain behaviors. Anexample is the optokinetic nystagmus test (Balkema G W et al., 1984,Invest Ophthalmol Vis Sci. 25:795-800; Mitchiner J C et al., 1976,Vision Res. 16:1169-71).

The present invention may also be used in combination with other formsof vision therapy known in the art to improve or restore vision. Forexample, the use of visual prostheses, which include retinal implants,cortical implants, lateral geniculate nucleus implants, or optic nerveimplants. Thus, in addition to genetic modification of surviving retinalneurons using the present methods, the subject being treated may beprovided with a visual prosthesis before, at the same time as, or afterthe molecular method is employed. The effectiveness of visualprosthetics can be improved with training of the individual, thusenhancing the potential impact of the CoChop transformation of patientcells as contemplated herein. Training methods, such as habituationtraining characterized by training the subject to recognize recognize(i) varying levels of light and/or pattern stimulation, and/or (ii)environmental stimulation from a common light source or object as wouldbe understood by one skilled in the art; and orientation and mobilitytraining characterized by training the subject to detect visually localobjects and move among said objects more effectively than without thetraining. In fact, any visual stimulation techniques that are typicallyused in the field of low vision rehabilitation are applicable here.

In some embodiments, use of different opsin genes in addition to themutant CoChop proteins of the present invention and targeted geneexpression may further increase light sensitivity or improve vision.Visual information is processed through the retina through two pathways:an ON pathway which signals the light ON, and an OFF pathway whichsignals the light OFF. The existence of the ON and OFF pathway isimportant for the enhancement of contrast sensitivity. The visual signalin the ON pathway is relay from ON-cone bipolar cells to ON ganglioncells. Both ON-cone bipolar cells and ON-ganglion cells are depolarizedin response to light. On the other hand, the visual signal in the OFFpathway is carried from OFF-cone bipolar cells to OFF ganglion cells.Both OFF-cone bipolar cells and OFF-ganglion cells are hypopolarized inresponse to light. Rod bipolar cells, which are responsible for theability to see in dim light (scotopic vision), are ON bipolar cells(depolarized in response to light). Rod bipolar cells relay the visionsignal through AII amacrine cells (an ON type retinal cells) to ON anOFF cone bipolar cells.

Accordingly, a dual rhodopsin system can be used to recapitulate the ONand OFF pathways integral to visual processing and acuity. Briefly, aCoChop protein of the present invention can be specifically targeted toON type retinal neurons (i.e., ON type ganglion cells and/or ON typebipolar cells), while a hypopolarizing light sensor (i.e., halorhodopsinor other chloride pump known in the art) can be targeted to OFF typeretinal neurons (i.e. OFF type ganglion cells and/or OFF type bipolarcells) to create ON and OFF pathways. The specific targeting topreferred cell subpopulations can be achieved through the use ofdifferent cell type-specific promoters. For example, CoChop expressionmay be driven by the mGluR6 promoter for targeted expression in ON-typeretinal neurons (i.e., ON type ganglion cells and/or ON type bipolarcells) while a hypopolarizing channel, such as halorhodopsin, expressionis driven by the NK-3 promoter for targeted expression in OFF-typeretinal neurons (i.e., OFF type ganglion cells and/or OFF type bipolarcells).

An alternative approach to restore ON and OFF pathways in the retina isachieved by, expressing a depolarizing light sensor, to rod bipolarcells or AII amacrine. In this approach, the depolarization of rodbipolar cells or AII amacrine cells can lead to the ON and OFF responsesat the levels of cone bipolar cells and the downstream retinal ganglioncells. Thus, the ON and OFF pathways that are inherent in the retina aremaintained.

Method of Delivery

In some aspects, the pharmaceutical composition is administered by anymethod known in the art to treat or prevent a particular disease ordisorder. In preferred embodiments, when treating ocular disorders thepharmaceutical composition is administered to intravitreal sites usingany direction method. In some cases, the delivery method may be byinjection, such as those described in US Pat Pub. No. 2010008170, whichis incorporated by reference in its entirety. In some cases, directadministration to the vitreous includes injection of a liquidpharmaceutical composition via syringe. In another example, directadministration may involve injection via a cannula or other suitableinstrument for delivery for a vector or recombinant virus. In otherexamples, direct administration may comprise an implant furthercomprising a suitable vector for delivery of transgenes such as mCoChop.In some cases the implant may be either directly implanted in or nearthe retina.

Generally, the vector can be delivered in the form of a suspensioninjected intraocularly (intravitreally). Specifically, the vector isinjected transclerally through the pars plana.

Definitions

The compositions and methods of this disclosure as described herein mayemploy, unless otherwise indicated, conventional techniques anddescriptions of molecular biology (including recombinant techniques),cell biology, biochemistry, immunochemistry and ophthalmic techniques,which are within the skill of those who practice in the art. Suchconventional techniques include methods for observing and analyzing theretina, or vision in a subject, cloning and propagation of recombinantvirus, formulation of a pharmaceutical composition, and biochemicalpurification and immunochemistry. Specific illustrations of suitabletechniques can be had by reference to the examples herein. However,equivalent conventional procedures can, of course, also be used. Suchconventional techniques and descriptions can be found in standardlaboratory manuals such as Green, et al., Eds., Genome Analysis: ALaboratory Manual Series (Vols. I-IV) (1999); Weiner, et al., Eds.,Genetic Variation: A Laboratory Manual (2007); Dieffenbach, Dveksler,Eds., PCR Primer: A Laboratory Manual (2003); Bowtell and Sambrook, DNAMicroarrays: A Molecular Cloning Manual (2003); Mount, Bioinformatics:Sequence and Genome Analysis (2004); Sambrook and Russell, CondensedProtocols from Molecular Cloning: A Laboratory Manual (2006); andSambrook and Russell, Molecular Cloning: A Laboratory Manual (2002) (allfrom Cold Spring Harbor Laboratory Press); Stryer, L., Biochemistry (4thEd.) W.H. Freeman, N.Y. (1995); Gait, “Oligonucleotide Synthesis: APractical Approach” IRL Press, London (1984); Nelson and Cox, Lehninger,Principles of Biochemistry, 3rd Ed., W.H. Freeman Pub., New York (2000);and Berg et al., Biochemistry, 5th Ed., W.H. Freeman Pub., New York(2002), all of which are herein incorporated by reference in theirentirety for all purposes.

As used herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Furthermore, to the extent that the terms “including”,“includes”, “having”, “has”, “with”, or variants thereof are used ineither the detailed description and/or the claims, such terms areintended to be inclusive in a manner similar to the term “comprising”.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another case includes from the one particular value and/or tothe other particular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about,” it will be understoodthat the particular value forms another case. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint. The term “about” as used herein refers to a range that is 15%plus or minus from a stated numerical value within the context of theparticular usage. For example, about 10 would include a range from 8.5to 11.5. The term “about” also accounts for typical error or imprecisionin measurement of values.

The term “retinal degenerative diseases” encompasses all diseasesassociated with photoreceptor degeneration. Retinal degenerativediseases include but are not limited to Retinitis Pigmentosa,age-related macular degeneration, Bardet-Biedel syndrome,Bassen-Kornzweig syndrome, Best disease, choroideremia, gyrate atrophy,Leber congenital amaurosis, Refsun syndrome, Stargardt disease or Ushersyndrome.

In the context of the invention, the term “treating” or “treatment”, asused herein, means reversing, alleviating, inhibiting the progress of,or preventing the disorder or condition to which such term applies, orone or more symptoms of such disorder or condition (e.g., retinaldegenerative diseases).

According to the invention, the term “patient” or “patient in needthereof”, is intended for a human or non-human mammal affected or likelyto be affected with a retinal degenerative disease.

As intended herein the expression “isolated nucleic acid” refers to anytype of isolated nucleic acid, it can notably be natural or synthetic,DNA or RNA, single or double stranded. In particular, where the nucleicacid is synthetic, it can comprise non-natural modifications of thebases or bonds, in particular for increasing the resistance todegradation of the nucleic acid. Where the nucleic acid is RNA, themodifications notably encompass capping its ends or modifying the 2′position of the ribose backbone so as to decrease the reactivity of thehydroxyl moiety, for instance by suppressing the hydroxyl moiety (toyield a 2′-deoxyribose or a 2′-deoxyribose-2′-fluororibose), orsubstituting the hydroxyl moiety with an alkyl group, such as a methylgroup (to yield a 2′-O-methyl-ribose).

The term “channelrhodopsin” refers to the subfamily of retinylideneproteins (rhodopsins) that function as light-gated ion channels. Someserve as sensory photoreceptors in unicellular green algae, controllingphototaxis: movement in response to light. Expressed in cells of otherorganisms, they enable light to control electrical excitability,intracellular acidity, calcium influx, and other cellular processes.They are larger than many other rhodopsins, with a 7 transmembrane (7TM)region and a long C-terminal extension. In algae they function as visualproteins directing the alga towards or away from a light source and tofind light conditions that are optimal for photosynthetic growth. The7TM region shows some homology to other microbial (procaryotic)rhodopsins functioning as light-driven pumps (bacteriorhodopsin,archeorhodopsin and halorhodopsin) or sensors. Term also includepolypeptides that are homologous to channelrhodopsin.

Two amino acid sequences or nucleic acid sequences are “substantiallyhomologous” or “substantially similar” when greater than 80%, preferablygreater than 85%, preferably greater than 90% of the amino acids ornucleic acid sequences are identical, or greater than about 90%,preferably greater than 95%, are similar (functionally identical). Todetermine the percent identity of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of a first amino acid ornucleic acid sequence for optimal alignment with a second amino ornucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences. In one embodiment, thetwo sequences are the same length. The determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. Preferably, the similar or homologous sequences areidentified by alignment using, for example, the GCG (Genetics ComputerGroup, Program Manual for the GCG Package, Version 7, Madison, Wis.)pileup program, or any of sequence comparison algorithms such as BLAST,FASTA, etc.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a “plasmid”, which refers to a circulardouble stranded DNA loop into which additional DNA segments can beligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors, expressionvectors, are capable of directing the expression of genes to which theyare operably linked.

Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference in theirentireties. All published foreign patents and patent applications citedherein are hereby incorporated by reference in their entireties. Allother published references, documents, manuscripts and scientificliterature cited herein are hereby incorporated by reference in theirentireties.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

This disclosure is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1—Creation and Analysis of Mutant CoChop Polypeptides

Channelrhodopsons (ChRs), such ChR2, are promising optogenetic lightsensors for vision restoration. A major obstacle for using ChR2 invision restoration is its low light sensitivity. We previously made morelight-sensitive ChR2s by optimizing its kinetics through site-directmutagenesis, including the most light-sensitive ChR2 mutant,ChR2-L132C/T159S. Recently, a number of ChR variants have been reportedby de novo transcriptome sequencing of algae (Klapoetke et al., 2014Nat. Methods 11(3): 338-46). We found that one of the variants, CoChR,displayed large photocurrent. In this invention, we made several highlylight-sensitive CoChR mutants (i.e. mutant CoChop) by optimizing itskinetics through site-direct mutagenesis. These mutants includeCoChR-L112C (SEQ ID NO: 3), CoChR-T139C (SEQ ID NO: 5), C68S/V69I (SEQID NO: 4), C68T/V69I (SEQ ID NO: 7), CoChR-T145A/S146A (SEQ ID NO: 6),CoChR-L112C/T139C (SEQ ID NO: 8), CoChR-L112C/H94E (SEQ ID NO: 9), andCoChR-L112C/H94E/K264T (SEQ ID NO: 10). CoChR and its mutants exhibit aslight red-shifted spectral curve than that of ChR2 with a peak spectrumat 480 nm (FIG. 1). The light sensitivity of CoChR mutants (as shown forCoChR-L112C) are much higher than that of the most light-sensitive ChR2mutant, ChR2-L132C/T159S, based on electrophysiology recordings in HEKcells (FIGS. 2-5) and multi-electrode array recordings from retinalneurons (FIG. 6), and optomotor behavioral tests from blind mice in vivo(FIG. 7). Furthermore, optomotor response can be observed forCoChR-L112C-expressing mice under ambient light conditions (FIG. 8). Inaddition, long-term stable expression of CoChR-L112C mutant was observedin retinal neurons (FIGS. 9A-9G).

1-24. (canceled)
 25. A method of treating an ocular disease or disorderby improving or restoring vision in a subject in need of such therapy,the method comprising administering to the subject a therapeuticallyeffective amount of a pharmaceutical composition comprising a nucleicacid encoding a polypeptide comprising the amino acid sequence of anyone of SEQ ID NOs: 3-10, wherein the nucleic acid is operably linked toa promoter and the polypeptide is expressed in a cell membrane of thesubject following administration.
 26. The method of claim 25, whereinthe nucleic acid is incorporated into a recombinant adeno-associatedvirus (rAAV) viral vector.
 27. The method of claim 26, wherein the rAAVis a recombinant form of an AAV selected from the group consisting ofAAV1, AAV2, AAV2.5, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10,AAV11, AAV12, rh10.
 28. The method of claim 27, wherein the AAV is AAV2.29. The method of claim 25, wherein the ocular disease or disorder isglaucoma, cataracts, corneal dystrophy, or keratoconus.
 30. The methodof claim 25, wherein the ocular disease or disorder is a retinaldisorder.
 31. The method of claim 30, wherein the retinal disorder iscongenital stationary night blindness, macular degeneration, congenitalcone dystrophies, or a retinitis-pigmentosa (RP)-related disorders. 32.The method of claim 31, wherein the macular degeneration is age-relatedmacular degeneration (AMD).
 33. The method of claim 31, wherein theretinal disorder is retinitis pigmentosa or diabetic retinopathy. 34.The method of claim 25, wherein the ocular disease or disorder is achildhood onset blinding disease.
 35. The method of claim 25, whereinsaid improving or restoring vision comprises any one or more ofincreasing light sensitivity, lowering a threshold light intensityrequired to elicit a photocurrent, increasing ion flux and/or protonflux, or increasing visual evoked potential in the visual cortex. 36.The method of claim 25, wherein the pharmaceutical composition isadministered to an eye of the subject by intravitreal injection,subretinal injection, injection via a cannula, or an implant.
 37. Themethod of claim 36, wherein the pharmaceutical composition isadministered to an eye of the subject by intravitreal or subretinalinjection.
 38. The method of claim 25, wherein the subject is a human.39. A method of treating macular degeneration in a human subject in needof such therapy, the method comprising administering to the subject atherapeutically effective amount of a pharmaceutical compositioncomprising a nucleic acid encoding a polypeptide comprising the aminoacid sequence of any one of SEQ ID NOs: 3-10, wherein the nucleic acidis operably linked to a promoter and the polypeptide is expressed in acell membrane of the subject following administration.
 40. The method ofclaim 39, wherein the nucleic acid is incorporated into a recombinantadeno-associated virus (rAAV) viral vector.
 41. The method of claim 40,wherein the rAAV is a recombinant form of an AAV selected from the groupconsisting of AAV1, AAV2, AAV2.5, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8,AAV9, AAV10, AAV11, AAV12, rh10.
 42. The method of claim 41, wherein theAAV is AAV2.
 43. The method of claim 39, wherein said improving orrestoring vision comprises any one or more of increasing lightsensitivity, lowering a threshold light intensity required to elicit aphotocurrent, increasing ion flux and/or proton flux, or increasingvisual evoked potential in the visual cortex.
 44. The method of claim40, wherein the pharmaceutical composition is administered to an eye ofthe subject by intravitreal injection, subretinal injection, injectionvia a cannula, or an implant.